Name: Video: Barwienie immunofluorescencyjne populacji neuronów w embrionalnym mysim przewodzie pokarmowym - Concept
Uploaded: 2025-03-04T15:34:58.000+00:00
Duration: 1 min 20 s
Description: 32 Views. Źródło: Barlow-Anacker, A. J., et al., Barwienie immunologiczne w celu wizualizacji rozwoju mysiego jelitowego układu nerwowego. J. Vis. Exp. (2015) Ten film demonstruje barwienie immunologiczne embrionalnego mysiego przewodu pokarmowego w celu wizualizacji i analizy rozmieszczenia populacji neuronów za pomocą immunofluorescencji i mikroskopii konfokalnej.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunostaining Protocol
<ol>
	<li>If samples have been stored in 30% sucrose, rinse them 3 times for 20 min in 1x PBS on a rocking platform.</li>
	<li>Place the GI tracts into the blocking solution on a rocking platform for 1h at RT.</li>
	<li>Remove the blocking solution and incubate the GI tracts with the appropriate amount of primary antibodies diluted in the blocking solution for either 4 hr at RT or O/N at 4 &#176;C on a rocking platform. Use a 1:1,000 dilution of human anti-Hu antibody (serum obtained from the patient), a 1:1,000 dilution of chicken anti-green fluorescent protein (GFP) antibody, and a 1:100 dilution of goat anti-ChAT antibody. 
	NOTE: We utilize a human anti-Hu antibody that was obtained locally from a patient; however, anti-Hu antibodies are commercially available, for example, mouse anti-Hu (use at 1:500 dilution).</li>
	<li>Rinse the GI tracts in 1x PBS 3 times for 5 min and then for 1 hr at RT on a rocking platform.</li>
	<li>Replace the 1x PBS with secondary antibodies diluted 1:500 in blocking solution on a rocking platform for either 4 hr at RT or O/N at 4 &#176;C. Use donkey anti-human amine-reactive dye such as Dylight, donkey anti-chicken Cy2 and donkey anti-goat Cy5. If using the mouse anti-Hu antibody, then use a 1:500 dilution of donkey anti-mouse amine-reactive dye 405. 
	NOTE: The intensity of the endogenous GFP and tdTomato expression and the clarity of immunostaining increase with developmental age. We typically immunostain embryonic guts individually in 0.2 ml tubes with 150 &#956;l of staining solution in order to reduce the volume of antibody used and also to ensure efficient staining of the tissue.</li>
	<li>Rinse the GI tracts in 1x PBS 3 times for 5 min and then for 1 hr at RT on a rocking platform.</li>
	<li>Place a few drops of fluorescence mounting medium with DAPI onto a glass slide. Immerse the GI tract into the fluorescence mounting medium and add a cover glass directly on top of the tissue. The fluorescence mounting medium -G is a water-soluble compound that provides a semi-permanent seal. 
	NOTE: Use fluorescence mounting medium such as Vectashield, a glycerol-based mounting medium that prevents antibody fading and photobleaching. Both of these products enable tissues to be stored for long periods of time at 4 &#176;C.</li>
	<li>Capture images of each fluorophore using a confocal microscope. Use the excitation wavelengths for fluorophores and filters employed in Table 1.</li>
	<li>Perform computer-aided image analysis using appropriate software depending on the type of confocal used to capture images.</li>
</ol>
Table 1: &#160;Confocal imaging excitation wavelengths, fluorophores, and filters. The excitation wavelengths, detectable fluorophores, and filters employed are presented. This information can be used in choosing combinations of secondary antibodies for multicolor immunofluoresence.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Excitation&#160;&#160;&#160; </td>
			<td colspan="2">Emission</td>
		</tr>
		<tr>
			<td>Laser Line</td>
			<td>Fluorophore Type</td>
			<td>Examples</td>
			<td>Photomultiplier Tube</td>
			<td>Filter Options</td>
		</tr>
		<tr>
			<td>408 nm</td>
			<td>Blue</td>
			<td>Alexa Fluor 405, Cascade Blue, Coumarin 30, DAPI, Hoechst, Pacific Blue, most quantum dots</td>
			<td>1</td>
			<td>BP 425-475 nm</td>
		</tr>
		<tr>
			<td>488 nm</td>
			<td>Green</td>
			<td>Alexa Fluor 488, ATTO 488, Calcein, Cy2, eGFP, FITC, Oregon Green, YO-PRO-1</td>
			<td>2</td>
			<td>BP 500-550 nm</td>
		</tr>
		<tr>
			<td>561 nm</td>
			<td>Red</td>
			<td>Alexa Fluor 546, 555, 568, and 594, Cy3, DiI, DsRed, mCherry, Phycoerythrin (PE), Propidium Iodine (PI), RFP, TAMRA, tdTomato, TRITC</td>
			<td>3</td>
			<td>BP 570-620 nm</td>
		</tr>
		<tr>
			<td>638 nm</td>
			<td>Far Red</td>
			<td>Alexa Fluor 633 and 647, Allophycocyanin (APC), Cy5, TO-PRO-3</td>
			<td>4</td>
			<td>BP 663-738 nm</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Oxoid</td>
			<td>BR0014G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher</td>
			<td>BP1605</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence scope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tubes</td>
			<td>VWR</td>
			<td>20170-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech, Birmingham, AL</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>16004-330</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>Nikon A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Elements</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

immunofluorescent staining of neuronal populations in the embryonic murine gastrointestinal tract

Source: Barlow-Anacker, A. J., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/52716">Immunostaining to Visualize Murine Enteric Nervous System Development.</a> J. Vis. Exp. (2015)
This video demonstrates the immunostaining of the embryonic murine gastrointestinal tract to visualize and analyze the neuronal population distribution using immunofluorescence and confocal microscopy.

Immunofluorescent Staining of Neuronal Populations in the Embryonic Murine Gastrointestinal Tract

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunostaining Protocol

	...

Take a fixed, transgenic embryonic mouse gastrointestinal tract at the desired developmental stage.
The gastrointestinal tract&#39;s enteric nervous system includes enteric neurons and cholinergic neurons expressing choline acetyltransferase.
These cholinergic neurons also express a fluorescent protein driven by the choline acetyltransferase promoter.
Incubate the tract with a non-ionic detergent and proteins to permeabilize cells and prevent non-specific antibody binding.
Replace with primary antibodies. Incubate for the antibodies to bind their target proteins in neurons, respectively.
Remove the unbound antibodies and wash with buffer.
Incubate with fluorophore-conjugated secondary antibodies to specifically bind the primary antibodies bound to target proteins.
Remove the excess antibodies and wash with buffer.
Immerse the labeled tract in mounting media containing a nuclear dye to stain the nuclei. Now, mount a coverslip.
Using confocal microscopy, visualize and analyze the distribution of cholinergic neurons in the gastrointestinal tract.

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Developmental and Pediatric Disorders

32 Views. Źródło: Barlow-Anacker, A. J., et al., Barwienie immunologiczne w celu wizualizacji rozwoju mysiego jelitowego układu nerwowego. J. Vis. Exp. (2015) Ten film demonstruje barwienie immunologiczne embrionalnego mysiego przewodu pokarmowego w celu wizualizacji i analizy rozmieszczenia populacji neuronów za pomocą immunofluorescencji i mikroskopii konfokalnej. 

Video: Barwienie immunofluorescencyjne populacji neuronów w embrionalnym mysim przewodzie pokarmowym - Concept

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.

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 ...

JoVE Journal

Developmental Biology

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 &#181;m (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY&#160;can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.

Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...

Immunofluorescent Staining of Neuronal Populations in the Embryonic Murine Gastrointestinal Tract

Transkrypcja

Immunofluorescent Staining of Neuronal Populations in the Embryonic Murine Gastrointestinal Tract