We are grateful to Chau-Ti Ting (Fly Core in Taiwan) for providing the monoclonal 4D9 antibody, Technology Commons (TechComm) of the College of Life Science NTU for confocal microscopy, Hsiao-Ling Lu for proofreading the manuscript, and Chen-yo Chung for helping filming. CC particularly thanks Charles E. Cook for providing strategic suggestions and for critical editing of the manuscript. This work was supported by the Ministry of Science and Technology (101-2313-B-002-059-MY3 and 104-2313-B-002-022-MY3 for GWL and CC), and the National Taiwan University (NTU-CESRP 101R4602D3 for CC; 103R4000 for GWL).

1. Culture of Aphids
NOTE: The laboratory strain of the parthenogenetic viviparous pea aphid A. pisum was originally collected in the central Taiwan and has been reared on host plants (the garden pea Pisum sativum or broad bean Vicia faba) under long-day photoperiod for more than 300 generations (one generation: ~10 days).
<ol>
	<li>Germination of Seeds &#160; &#160; &#160;
	<ol>
		<li>Soak the seeds of host plants in tap water for 3-5 days at RT. Refill with fresh water once per day. 
		NOTE: Alternatively, putting seeds of host plants straight into moist soil can induce germination as well.</li>
		<li>Grow 10 germinating seeds in a small pot (9 cm diameter x 7 cm tall) with soil in the growth chamber under photoperiod 16 hr light/8 hr dark at 20 &#176;C.</li>
		<li>About 10 days after growth begins, transfer aphids onto plants whose height is more than 8 cm. &#160;</li>
	</ol>
	</li>
	<li>Transfer of Aphids
	<ol>
		<li>Keep each pot of plants within a 1 L glass beaker, transfer 8 adult aphids onto plants using a paintbrush, and then seal the beaker with an air-permeable cover such as gauze mesh to prevent aphids from escaping.</li>
		<li>Incubate aphids in a growth chamber under photoperiod 16 hr light/8 hr dark at 20 &#176;C. Water each plant pot of plants once every day.</li>
		<li>For obtaining the next generation of aphids, reiterate steps 1.1.3-1.2.2 ten days after the primary aphid transfer.</li>
	</ol>
	</li>
</ol>
2. Dissection and Fixation of Ovaries
<ol>
	<li>Freshly prepare 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS) as the fixation buffer.</li>
	<li>Fill one well of a spot plate with PFA (about 500 &#181;l), place the plate under a stereo microscope at low magnification, and submerge an adult aphid within the PFA for dissection.</li>
	<li>Dissect ovaries by holding the head and abdomen with one set of forceps, cutting open the dorsal cuticle of the abdomen, and dragging the ovaries away from the abdominal cavity.</li>
	<li>Fix three pairs of ovaries in a 1.5 ml tube containing 1 ml of PFA at RT for 20 min.</li>
	<li>Decant fixation buffer with a transfer pipette (either glass or disposable) and then wash ovaries with 0.2% TritonX-100 in 1x PBS (PBST) 3 times for 10 min each. Mild shaking on a mixer/rotator (rotation angle: 60&#176; (F60)/rotation speed: 8 RPM) is recommended for both fixation and washing.</li>
</ol>
3. Treatment with Proteinase K (PK) to Increase the Permeability of Embryonic Tissues
NOTE: PK treatment is applied to embryos from germ band extension onward (stage 11 of development). For younger embryos, this step is optional.
<ol>
	<li>Serially dilute the stock solution of PK (10 mg/ml) with 1x PBS to the working concentration 1 &#181;g/ml.</li>
	<li>Incubate ovaries with 1 &#181;g/ml of PK (about 500 &#181;l) for 10 mins with mild shaking.</li>
	<li>Decant PK solution and then wash the ovaries with 700 &#181;l of Glycine (2 mg/ml) 3 times for 5 mins each.</li>
	<li>Wash ovaries with 0.2% PBST twice for 10 min each.</li>
	<li>Fix ovaries again with fixation buffer for 15 min at RT with mild shaking.</li>
	<li>Discard supernatant and wash ovaries with 0.2% PBST twice for 10 min each.</li>
</ol>
4. Methanol Incubation for Suppressing the Endogenous peroxidase (POD) activity
NOTE: Methanol incubation is not applied to embryos subjected to Phalloidin staining or antibody epitopes that are methanol sensitive.
<ol>
	<li>Serially dehydrate ovaries with different percentage of methanol in 0.2% PBST (v/v: 1:3, 1:1, 3:1) by incubating ovaries at each concentration of methanol solution for 10 min with mild agitation.</li>
	<li>Dehydrate ovaries with 100% methanol for 1 hr at RT with mild shaking. 
	NOTE: Satisfactory results of staining could still be obtained from aphid tissues that were stored in 100% methanol at -20 &#176;C for one month.</li>
	<li>Serially rehydrate ovaries with different percentage of methanol in 0.2% PBST (v/v: 3:1, 1:1, 1:3) by incubating ovaries at each concentration of methanol solution for 10 min with mild agitation.</li>
</ol>
5. Antibody Staining
<ol>
	<li>Dilute the 10x blocking solution from the DIG-based buffer set (DIG-B) to 1x. 
	NOTE: The 1x DIG-B blocking solution is more effective for reducing the staining background than the standard blocking reagent composed of 5% (v/v) normal goat serum (NGS) and 0.5% (v/v) bovine serum albumin (BSA) in 0.2% PBST.</li>
	<li>Incubate the ovaries with the 1x DIG-B blocking solution for 2.5 to 4 hr at RT or O/N at 4 &#176;C with mild shaking. 
	NOTE: For three pairs of ovaries in a 1.5 ml tube, 200 &#181;l is the minimum volume for sample blocking and antibody staining.</li>
	<li>Decant supernatant and replace with fresh 1x DIG-B blocking solution containing primary antibody at appropriate dilution ratio. Stain the ovaries for 4 hr at RT or O/N at 4 &#176;C with mild shaking. 
	NOTE: For the experiment described here, use the following optimal dilutions of primary antibodies: (1) ApVas1 antibody: 1:500 for chromogenic staining, 1:50 for immunofluorescence staining; (2) anti-&#945; tubulin antibody: 1:500; (3) 4D9 monoclonal antibody: 1:25.</li>
	<li>Wash ovaries with 0.2% PBST 4 times for 15 min each.</li>
	<li>Incubate ovaries with 1x DIG-B blocking solution for 1 hr at RT with mild shaking.</li>
	<li>Decant supernatant and replace with fresh 1x DIG-B blocking solution containing secondary antibody at appropriate dilution ratio. Stain the ovaries for 4 hr at RT or O/N at 4 &#176;C with mild shaking.
	<ol>
		<li>Use the following dilution ratios of secondary antibodies: (1) For immunofluorescence staining: 1:500 for Alexa Fluor 633 goat anti-rabbit IgG or Alexa Fluor 488 goat anti-mouse IgG; (2) For chromogenic staining: 1:200 for biotinylated goat anti-rabbit IgG. 
		NOTE: The biotinylated secondary antibody is applied for interacting with the avidin-biotin-complex (ABC) in the substrate-based (chromogenic) detection, through which the staining signals are significantly amplified.</li>
		<li>For immunofluorescence staining, carry out staining in the dark because the secondary antibody is light sensitive.</li>
	</ol>
	</li>
	<li>Wash off secondary antibody with 0.2% PBST 4 times for 10 min each.</li>
</ol>
6. Nuclear and F-actin Staining
NOTE: This is only applied for immunofluorescence staining.
<ol>
	<li>Stain ovaries with 0.2% PBST containing DAPI (2 ng/&#181;l) and Phalloidin-TRITC (100 nM) for 2 hr at RT in dark.</li>
	<li>Wash ovaries with 0.2% PBST 4 times for 10 min each.</li>
	<li>Incubate ovaries with the mounting medium O/N at 4 &#176;C with mild shaking.</li>
</ol>
7. Signal Development
NOTE: This is only applied for chromogenic staining.
<ol>
	<li>Prepare 100 &#181;l of reagent avidin-biotin-complex (ABC) for enhancing the signals by adding 1 &#181;l of Reagent A (avidin) in 98 &#181;l of 0.2% PBST, mixing thoroughly via gentle pipetting, and adding 1 &#181;l of Reagent B (biotin conjugated with horseradish peroxidase), followed by immediate mixing. Incubate the mixture for 30 min at RT with mild shaking.</li>
	<li>Incubate ovaries (including the disassociated egg chambers) in the mixture of reagents A and B for 30 min at RT with mild shaking.</li>
	<li>Wash off the mixture of reagents A and B with 0.2% PBST 4 times for 10 min each.</li>
	<li>Transfer ovaries submerged in PBST to a well on the spot plate with a plastic dropper.</li>
	<li>Prepare substrate solution of 3,3&#39;-Diaminobenzidine (DAB): dissolve one DAB tablet plus another one containing urea hydrogen peroxide in 1 ml of ddH2O via vigorous vortexing for 1 min. 
	NOTE: DAB, a precipitating substrate of peroxidase, is a popular chromogen for immunostaining. It produces a brown and insoluble precipitate after being oxidized by the peroxidase. Weak signals can be enhanced by adding nickel chloride to the substrate solution (final concentration 0.05-0.08 %).</li>
	<li>Remove the PBST solution remaining in the well and refill with 100 &#181;l of the DAB substrate solution for signal development.</li>
	<li>Monitor intensity of signals under a stereo microscope at low magnification.</li>
	<li>Stop reactions by removing the DAB solution, followed by refilling with 1x PBS immediately. Repeat the wash twice.</li>
	<li>Transfer ovaries back to the 1.5 ml tube and then wash with 1x PBS or PBST twice for 15 min each.&#160;</li>
	<li>Incubate ovaries in a glycerol-based mounting medium (70% glycerol) O/N at 4 &#176;C with mild shaking.</li>
</ol>
8. Mounting the Aphid Embryos
NOTE: The thickness of aphid embryos varies between stages of development. Mounting strategies are thus modified to fit early (germaria and stages 0-10), mid (stages 11-18), and late embryos (stages 19-20), which are demonstrated in Figure 2B-D. Embryonic staging followed Miura et al.12
<ol>
	<li>Transfer ovaries together with mounting medium to the cell tray with a plastic dropper and observe samples under a stereo microscope at low magnification.</li>
	<li>Cut the calyces associated with the lateral oviduct using insect pins and then transfer an isolated ovariole to an empty glass well with a dropper. Make up the final volume to 50-100 &#181;l using mounting medium.</li>
	<li>Transfer an ovariole onto the slide with a glass dropper and then dissect egg chambers using insect pins. 
	NOTE: For embryos older than stage 6 of development, separation of egg chambers is suggested; for germaria and the first two egg chambers younger than stage 6, separation is optional.</li>
	<li>Relocate a dissected egg chamber to a clean slide using a glass dropper.</li>
	<li>Put a coverslip (size: 22 x 22 mm) over the dissected germaria or egg chambers (containing embryos at stages 1-10 of development) slowly to avoid bubbles.
	<ol>
		<li>Mount egg chambers (containing embryos at stages 11-18 of development) on a slide with one-sided coverslip bridge and place another coverslip (size: 18 x 18 mm) on top of the sample.</li>
		<li>Mount egg chambers (containing embryos older than stage 19 of development) on a slide with double-sided coverslip bridge and place another coverslip (size: 18 x 18 mm) on top of the sample.</li>
	</ol>
	</li>
	<li>Fill the space beneath the top coverslip with mounting media to avoid drying the sample.</li>
	<li>Mildly roll the embryo by sliding the coverslip to obtain the right orientation for observation.</li>
	<li>Seal around the edge of coverslips (including the bridge coverslips) with nail polish.</li>
</ol>
9. Imaging Analysis
<ol>
	<li>Photograph differential interference contrast (DIC) images of whole-mount embryos with a compound microscope equipped with DIC optics and a dry objective lens (10X, 20X, 40X) connected to a camera. Install the software for image transfer between the camera and computer using manufacturer&#8217;s instructions.</li>
	<li>Acquire projections of the fluorescently labeled embryos with a laser-scanning confocal microscope14. Follow the manufacturer&#39;s instructions for photographing, z-stacking, and 3D projecting with imaging software.
	<ol>
		<li>Turn the slide upside down, find the mounted sample with 10X objective, and circle the sample area with a fine oiled-based pen. 
		NOTE: This makes identifying the sample easier when searching for it through objectives of a confocal microscope.</li>
		<li>Add a drop of oil on the top of the coverslip area whose opposite side is labeled as described in 9.2.1.</li>
	</ol>
	</li>
	<li>Find the focal plane at 40X oil-immersion objective then change to a 63X oil-immersion objective. Manually move the fine focus control up and down to capture the best focal plane. 
	NOTE: For observing structural details of germaria and early embryos, we suggest using 40X objectives or those with higher magnification.</li>
	<li>Scan the embryo in different excitation channels and obtain a z-stack image. 
	NOTE: For pea aphid tissues, reduce thickness of each optical section down to 1.5 &#181;m or less.</li>
</ol>

<ol>
	<li>Blackman, R. L., Minks, A. K., Harrewijn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproduction,+cytogenetics+and+development.">Reproduction, cytogenetics and development.</a> Aphids: their biology, natural enemies. , 163-195 (1987).</li><li>Davis, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclical+parthenogenesis+and+viviparity+in+aphids+as+evolutionary+novelties.">Cyclical parthenogenesis and viviparity in aphids as evolutionary novelties.</a> J. Exp. Zool. 318, 448-459 (2012).</li><li>The International Aphid Genomics Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequence+of+the+pea+aphid+Acyrthosiphon+pisum.">Genome sequence of the pea aphid Acyrthosiphon pisum.</a> PLoS Biol. 8 (2), e1000313 (2010).</li><li>Abzhanov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+we+there+yet?+Tracking+the+development+of+new+model+systems.">Are we there yet? Tracking the development of new model systems.</a> Trends Genet. 24 (7), 353-360 (2008).</li><li>Chang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apvasa+marks+germ-cell+migration+in+the+parthenogenetic+pea+aphid+Acyrthosiphon+pisum+(Hemiptera:+Aphidoidea)).">Apvasa marks germ-cell migration in the parthenogenetic pea aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea)).</a> Dev. Genes Evol. 217 (4), 275-287 (2007).</li><li>Chang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+identification+of+gene+transcripts+in+aphids:+Protocols+and+evaluation+of+probe+accessibility.">Whole-mount identification of gene transcripts in aphids: Protocols and evaluation of probe accessibility.</a> Arch. Insect Biochem. 68 (4), 186-196 (2008).</li><li>Chung, C. Y., Cook, C. E., Lin, G. W., Huang, T. Y., Chang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+protocols+for+whole-mount+fluorescent+in+situ+hybridization+(FISH)+in+the+pea+aphid+Acyrthosiphon+pisum:+a+comprehensive+survey+and+analysis.">Reliable protocols for whole-mount fluorescent in situ hybridization (FISH) in the pea aphid Acyrthosiphon pisum: a comprehensive survey and analysis.</a> Insect Sci. 21 (3), 265-277 (2014).</li><li>Mutti, N. S., Park, Y., Reese, J. C., Reeck, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+knockdown+of+a+salivary+transcript+leading+to+lethality+in+the+pea+aphid,+Acyrthosiphon+pisum.">RNAi knockdown of a salivary transcript leading to lethality in the pea aphid, Acyrthosiphon pisum.</a> J. Insect Sci. 6, 38 (2006).</li><li>Jaubert-Possamai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+knockdown+by+RNAi+in+the+pea+aphid+Acyrthosiphon+pisum.">Gene knockdown by RNAi in the pea aphid Acyrthosiphon pisum.</a> BMC Biotechnol. 7, 63 (2007).</li><li>Sapountzis, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insight+into+the+RNA+interference+response+against+cathepsin-L+gene+in+the+pea+aphid,+Acyrthosiphon+pisum:+Molting+or+gut+phenotypes+specifically+induced+by+injection+or+feeding+treatments.">New insight into the RNA interference response against cathepsin-L gene in the pea aphid, Acyrthosiphon pisum: Molting or gut phenotypes specifically induced by injection or feeding treatments.</a> Insect Biochem. Mol. Biol. 51, 20-32 (2014).</li><li>Braendle, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+origin+and+evolution+of+bacteriocytes+in+the+aphid-Buchnera+symbiosis.">Developmental origin and evolution of bacteriocytes in the aphid-Buchnera symbiosis.</a> PLoS Biol. 1 (1), e21 (2003).</li><li>Miura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+parthenogenetic+and+sexual+embryogenesis+of+the+pea+aphid+Acyrthosiphon+pisum+(Hemiptera:+Aphidoidea).">A comparison of parthenogenetic and sexual embryogenesis of the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea).</a> J. Exp. Zool. 295 (1), 59-81 (2003).</li><li>Chang, C., Lee, W. C., Cook, C. E., Lin, G. W., Chang, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-plasm+specification+and+germline+development+in+the+parthenogenetic+pea+aphid+Acyrthosiphon+pisum:+Vasa+and+Nanos+as+markers.">Germ-plasm specification and germline development in the parthenogenetic pea aphid Acyrthosiphon pisum: Vasa and Nanos as markers.</a> Int. J. Dev. Biol. 50 (4), 413-421 (2006).</li><li>Koshy, A. A., Cabral, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-D+imaging+and+analysis+of+neurons+infected+in+vivo+with+Toxoplasma+gondii.">3-D imaging and analysis of neurons infected in vivo with Toxoplasma gondii.</a> J. Vis. Exp. (94), e52237 (2014).</li><li>Brisson, J. A., Stern, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pea+aphid,+Acyrthosiphon+pisum:+an+emerging+genomic+model+system+for+ecological,+developmental+and+evolutionary+studies.">The pea aphid, Acyrthosiphon pisum: an emerging genomic model system for ecological, developmental and evolutionary studies.</a> Bioessays. 28 (7), 747-755 (2006).</li><li>Tautz, D., Pfeifle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-radioactive+in+situ+hybridization+method+for+the+localization+of+specific+RNAs+in+Drosophila+embryos+reveals+translational+control+of+the+segmentation+gene+hunchback.">A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback.</a> Chromosoma. 98 (2), 81-85 (1989).</li><li>Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-situ+hybridization:+an+improved+whole-mount+method+for+Xenopus+embryos.">In-situ hybridization: an improved whole-mount method for Xenopus embryos.</a> Method. Cell Biol. 36, 685-695 (1991).</li><li>Wolff, C., Sommer, R., Schroder, R., Glaser, G., Tautz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+and+divergent+expression+aspects+of+the+Drosophila+segmentation+gene+hunchback+in+the+short+germ+band+embryo+of+the+flour+beetle+Tribolium.">Conserved and divergent expression aspects of the Drosophila segmentation gene hunchback in the short germ band embryo of the flour beetle Tribolium.</a> Development. 121 (12), 4227-4236 (1995).</li><li>Jowett, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+in+situ+hybridization+techniques+in+zebrafish.">Double in situ hybridization techniques in zebrafish.</a> Methods. 23 (4), 345-358 (2001).</li><li>Lin, G. W., Cook, C. E., Miura, T., Chang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posterior+localization+of+ApVas1+positions+the+preformed+germ+plasm+in+the+sexual+oviparous+pea+aphid+Acyrthosiphon+pisum.">Posterior localization of ApVas1 positions the preformed germ plasm in the sexual oviparous pea aphid Acyrthosiphon pisum.</a> Evodevo. 5, 18 (2014).</li><li>Straus, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+peroxidase+by+methanol+and+by+methanol-nitroferricyanide+for+use+in+immunoperoxidase+procedures.">Inhibition of peroxidase by methanol and by methanol-nitroferricyanide for use in immunoperoxidase procedures.</a> J. Histochem. Cytochem. 19 (11), 682-688 (1971).</li></ol>

The authors have nothing to disclose.

We identified optimal conditions critical to successful immunostaining in the pea aphid A. pisum, an emerging model organism for genomic and developmental studies3,15. Optimized conditions for increasing tissue permeability and reducing background staining enhanced the intensity and specificity of signals. They differ from standard protocols for immunostaining in other animal models in the steps for creating pores in cell membranes and blocking non-specific antibody binding.
<p class="jov...

Aphids are hemipteran insects with small (1-10 mm) soft bodies. They feed on plants by sucking phloem sap with piercing mouthparts. Additionally, they rely on an obligate endosymbiotic bacterium, Buchnera aphidicola, to synthesize essential amino acids that are deficient in the phloem sap diet. Aphids have a complex life history that includes parthenogenetic viviparous reproduction during spring and summer long-day photoperiods and sexual oviparous reproduction triggered by short-day photoperiods during which they lay a limited number of overwintering eggs1,2. In spring these eggs hatch to produce the first generation of all-female aphids (fundatrices), following many rounds of parthenogenetic reproduction until autumn. The cyclical parthenogenesis in aphids, where asexual and sexual phases alternate in the annual life cycle, has been regarded as an evolutionary novelty1,2. In the parthenogenetic viviparous aphids, embryogenesis takes place within egg chambers of the ovarian tubules (ovarioles). By contrast, sexual oviparous embryos develop in the fertilized eggs. Apart from reproductive plasticity, aphids can display transgenerational wing polyphenism: in response to overcrowding signals and predator threats, the unwinged asexual females can viviparously produce winged offspring for long-distance migration. Publication of the genome sequence of the pea aphid Acyrthosiphon pisum-the first genome sequence for a basal hemimetabolous insect-allows further exploration of reproductive plasticity, wing polyphenism, and other features including insect-plant interactions, viral vectoring and symbiosis in aphids on a molecular basis3.
In addition to the sequenced genome, tools for characterizing gene expression and function are required for promoting the pea aphid as a mature model organism4. We have described robust protocols of whole-mount in situ hybridization for detecting expression of mRNA in aphid embryos5-7. RNA interference (RNAi) via double-stranded RNA injection and feeding has been used for gene silencing in aphid nymphs and adults, but stable conditions for gene knockdown in the embryos have not yet been reported8-10. Immunostaining, an antibody-based approach that can detect protein expression in samples before and after RNAi knockdown, has been performed on pea aphid embryos11-13. However, increase of tissue permeability and elimination of background staining are as yet unsatisfactory using standard protocols for immunostaining in the asexual viviparous embryos of the pea aphid. For example, we found that penetration of antibody to the tissues decreased in gastrulating embryos (stages 8-10) and that embryos with morphologically identifiable limb buds (stages 13-14) were barely permeable to antibody. In addition, background staining was visualized in the asexual viviparous pea aphid embryos stained using antibody against the germline marker Vasa as well as that against the Engrailed/Invected protein expressed in the embryonic segments12,13. Actually background staining was still clearly visible in embryos stained with the secondary antibody alone.
In order to increase permeability without damaging integrity of aphid tissues, we carefully titrated the concentration of proteinase K and determined optimal conditions for tissue digestion on aphid embryos. In order to avoid non-specific staining in the pea aphid, we searched for compounds that could effectively block embryos and suppress activity of endogenous peroxidase (POD), an enzyme employed for amplifying signals during immunostaining. A blocking reagent provided by a Digoxigenin (DIG)-based buffer set, rather the traditionally used normal goat serum (NGS)/bovine serum albumin (BSA), significantly reduced background staining. Moreover, methanol was found to inhibit the endogenous POD activity more effectively than hydrogen peroxide (H2O2). Details regarding these aphid-specific conditions for immunostaining on embryos will be described in the following sections.

<table><tbody><tr><td>30% hydrogen perxidase (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>18304</td><td>For bleaching endogenous peroxidase of embryos.</td></tr><tr><td>4&amp;rsquo;6-diamidino-2-phenyl-indole dihydrochloride (DAPI)</td><td>Sigma-Aldrich</td><td>D9542</td><td>For labeling the double-strand DNA. TOXIC. Wear gloves, dispense into small aliquots and feeze to minimize exposure</td></tr><tr><td>4D9 anti-engrailed/invected mouse monoclonal antibody</td><td>Developmental studies hybridoma bank</td><td>AB_528224</td><td>For labeling the developing segments of embryo.</td></tr><tr><td>ApVas1 rabbit polyclonal antibody</td><td>Our laboratory</td><td>N/A</td><td>For labeling the aphid germline specific Vas1 protein in embryos. This antibody was made by our laboratory.&amp;nbsp;</td></tr><tr><td>Austerlitz Insect pins</td><td>ENTOMORAVIA</td><td>N/A</td><td>For seperating each egg chambers from an ovariole. Size 000, BLACK ENAMELLED.&amp;nbsp;</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>9048-46-8</td><td>For blocking the non-specific binding of antibodies.</td></tr><tr><td>Camera connted to compound microscope</td><td>Canon</td><td>EOS 5D</td><td>For photographing of aphid embryos.</td></tr><tr><td>Colorimetric 8 cell tray</td><td>Kartell Labware</td><td>357</td><td>For developing the staining signal of aphid embryos. Diameter 95 x 57 mm, cell depth 2 mm.</td></tr><tr><td>Compound microscope with DIC optics</td><td>Leica Microsystems</td><td>DMR</td><td>For photographing of aphid embryo mounted on the slides.</td></tr><tr><td>DIG Wash and Block Buffer Set</td><td>Sigma-Aldrich (Roche)</td><td>11585762001</td><td>For blocking the non-specific binding of antibodies. We diluted the 10x Blocking solution into 1x as blocking reagent.</td></tr><tr><td>Forceps</td><td>Ideal-tek</td><td>N/A</td><td>For dissection of ovaries from aphids. Manufacturer part No 5: 5 SA.&amp;nbsp;</td></tr><tr><td>Glass dropper</td><td>N/A</td><td>N/A</td><td>For transfering ovaries of aphids from the splot plate to tubes. 150 mm of total length and 5 3/4&quot; of tip length.</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td>For clearing of aphid embryos and mounting.</td></tr><tr><td>Glycine</td><td>Bioshop</td><td>N/A</td><td>For blocking the enzyme activity of proteinase K (PK).</td></tr><tr><td>Goat anti-mouse IgG conjugated Alexa Fluor 488</td><td>Invitrogen</td><td>A11017</td><td>For detection of primary antibody from mouse.</td></tr><tr><td>Goat anti-rabbit IgG conjugated Alexa Fluor 633</td><td>Invitrogen</td><td>A21072</td><td>For detection of primary antibody from rabbit.</td></tr><tr><td>Intelli mixer</td><td>ELMI laboratory equipment</td><td>RM-2M</td><td>For improving the thorughly reaction of reagents with ovaries.</td></tr><tr><td>Laser-scanning confocal microscopy</td><td>Leica Microsystems</td><td>SP5</td><td>For photographing of aphid embryo mounted on the slides with florecent tag.</td></tr><tr><td>methanol&amp;nbsp;</td><td>Burdick and Jackson</td><td>AH2304</td><td>For bleaching endogenous peroxidase of embryos.</td></tr><tr><td>Microscope slides&amp;nbsp;</td><td>Thermo scientific</td><td>10143560</td><td>For mounting of embryos. SUPERFROST ground edges, ca./env. 76 x 26 mm.</td></tr><tr><td>Microscope Cover glasses</td><td>Marienfeld</td><td>0101030</td><td>For mounting embryos on the slides. Size 18 x 18 mm. Thickness No. 1 (0.13 to 0.16 mm)</td></tr><tr><td>Microscope Cover glasses</td><td>Marienfeld</td><td>0101050</td><td>For mounting embryos on the slides. Size 22 x 22 mm. Thickness No. 1 (0.13 to 0.16 mm)</td></tr><tr><td>mouse monoclonal anti-alphaTubulin antibody (DM1A)</td><td>Santa Cruz Biotechnology</td><td>sc-32293</td><td>For labeling the distrbution of alpha-tubulin of cells.</td></tr><tr><td>Nail polish</td><td>N/A</td><td>N/A</td><td>For sealing the coverslips on the slides.</td></tr><tr><td>Normal goat serum (NGS)</td><td>Sigma-Aldrich</td><td>G9023</td><td>For blocking the non-specific binding of antibodies.</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>VWR</td><td>MK262159</td><td>For fixation of aphid ovaries.</td></tr><tr><td>Phalloidin-TRITC</td><td>Sigma-Aldrich</td><td>P1951</td><td>For labeling the distrbution of F-actin on embryos.</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>N/A</td><td>N/A</td><td>As isotonic solution for aphid ovaries.</td></tr><tr><td>Plastic dropper</td><td>N/A</td><td>N/A</td><td>For transfering ovaries of aphids from tube to cell tray. Size 3 ml.&amp;nbsp;</td></tr><tr><td>Proteinase K</td><td>Merck</td><td>124678</td><td>For creating pores (punching) on the cell membrane and facilitating the access of antibodies.&amp;nbsp;</td></tr><tr><td>Pyrex spot plate</td><td>N/A</td><td>N/A</td><td>For dissection and color reactions of aphid ovaries under microscopy. Each cavity with diameter 22 mm and&amp;nbsp; depth 7 mm.</td></tr><tr><td>SIGMAFAST 3,3&amp;rsquo;-diaminobenzidine (DAB) tablets&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D4168</td><td>For developing of substrated signals. Also called DAB peroxidase substrate tablet set.</td></tr><tr><td>Stereo microscope</td><td>Leica Microsystems</td><td>EZ4</td><td>For dissection and observation of aphid ovaries.</td></tr><tr><td>Triton-X 100</td><td>Sigma-Aldrich</td><td>T8787</td><td>For creating pores (punching) on the cell membrane.&amp;nbsp;</td></tr><tr><td>VECTASHIELD Elite ABC Kit (Rabbit IgG)</td><td>Vector laboratories</td><td>PK-6101</td><td>For enhancement of the signal. Including of biotinylated goat anti-rabbit IgG secondary antibody, A and B reagents.</td></tr><tr><td>VECTASHIELD mounting medium</td><td>Vector laboratories</td><td>H1000</td><td>For clearing of aphid embryos and mounting.</td></tr></tbody></table>

identification of critical conditions for immunostaining in the pea aphid embryos: increasing tissue permeability and decreasing background staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 &#181;g/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

In this study, we performed whole-mount immunostaining on embryos of asexual pea aphids (Figure 1A). These females produce offspring parthenogenetically and viviparously. These female embryos develop within egg chambers of the ovarian tubules (ovarioles) (Figure 1B and Figure 2A). Before microscopy, the dissected ovarioles are the staining targets; however, separation of egg chambers is required for observation of embryos under a microsco...

Watch this Scientific Journal Video about Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining at JoVE.c

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

identification-critical-conditions-for-immunostaining-pea-aphid

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.9K Views.  National Taiwan University. A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

Video: Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Biology

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 &#176;C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules.

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model.

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

Summary

Explore More Videos

Chapters in this video

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

Method for Whole Mount Antibody Staining in Chick

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Wholemount In Situ Hybridization for Astyanax Embryos

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity