eoe sub articles

EoE Sub Articles

1. SARS-CoV-2 RNA-FISH in Vero cells cultured on coverslips
DAY 1 
<ol>
	<li>Fixing and permeabilizing cells&#8203;
	<ol>
		<li>Fix infected cells with 3.7% w/v PFA solution for 1 h at RT.</li>
		<li>Aspirate the 3.7% PFA solution, and wash the cells twice using 1x PBS.</li>
		<li>Permeabilize the cells with PBST solution for 10 min at RT with agitation.</li>
		<li>Aspirate the PBST, and wash the cells twice with 1x PBS.</li>
	</ol>
	</li>
	<li>Detection
	<ol>
		<li>Aspirate the 1x PBS solution, and wash the cells twice with 2x SSC at RT.</li>
		<li>Aspirate the 2x SSC solution, and prehybridize the samples by adding at least 300 &#181;L of probe hybridization buffer. Cover the wells containing the cells, and incubate at 37 &#176;C for 30 min.</li>
		<li>Prepare the probe solution by adding 1.2 pmol of probe mixture to the probe hybridization buffer.
		<ol>
			<li>Use 1.2 &#181;L of the 1 &#181;M probe stock to prepare 300 &#181;L of working stock.</li>
		</ol>
		</li>
		<li>Remove the prehybridization solution, and transfer the coverslips to a humidified chamber.
		<ol>
			<li>Pipette 30-50 &#181;L of probe solution onto parafilm to form individual droplets.</li>
		</ol>
		</li>
		<li>Incubate the samples overnight (12-18 h) at 37 &#176;C.</li>
	</ol>
	</li>
</ol>
DAY 2
<ol>
	<li>Transfer the coverslips back into a 12-well plate, and remove excess probe solution by washing for 4 x 5 min with 400 &#181;L of probe wash buffer at 37 &#176;C. 
	NOTE: As an alternative to placing 30-50 &#181;l aliquots onto parafilm and under coverslips for incubation, add 300 &#181;L of probe solution directly to coverslips in a 12-well plate. This procedure is simpler but requires substantial amounts of reagents. Heat the probe wash buffer to 37 &#176;C before use. Calculate the amount of buffer needed, and transfer it to a 15 mL conical centrifuge tube.</li>
	<li>Wash samples for 2 x 5 min with 5x SSCT at RT.</li>
	<li>Replace the 5x SSCT solution with 1x PBS, and store the samples at 4 &#176;C until amplification.</li>
</ol>
3. Amplification&#8203;
<ol>
	<li>Remove the 1x PBS solution from the wells, and add at least 300 &#181;L of amplification buffer to each well. Incubate the samples for 30 minutes at RT.</li>
	<li>Prepare each HCR hairpin (h1 and h2) by snap-cooling the desired volume in separate tubes.
	<ol>
		<li>To prepare 300 &#181;L of amplification solution, use 18 pmol of each hairpin (e.g., for 300 &#181;L, use 6 &#181;L of a 3 &#181;M stock hairpin solution).</li>
		<li>Transfer the hairpin solution into tubes.</li>
		<li>Incubate at 95 &#176;C for 90 s.</li>
		<li>Cool to RT for 30 min in the dark.</li>
	</ol>
	</li>
	<li>Prepare a hairpin mixture by adding the snap-cooled &#34;h1&#34; and &#34;h2&#34; hairpins to the amplification buffer.</li>
	<li>Place drops of 30-50 &#181;L of hairpin mixture onto parafilm.</li>
	<li>Incubate the samples overnight (12-18 h) in the dark at RT.</li>
</ol>
DAY 3
<ol start="6">
	<li>Transfer the coverslips back into the 12-well plate, and remove excess hairpins by washing for 5 x 5 min with 5x SSCT at RT with agitation.</li>
	<li>Aspirate the 5x SSCT buffer, and replace it with 1x PBS. 
	NOTE: If required, use the RNA-FISH samples in a standard immunofluorescence assay, followed by staining of nuclei (see section 1.4).</li>
</ol>
4. Nuclear staining and slide mounting&#8203;
<ol>
	<li>Aspirate the 1x PBS solution, and replace it with 4&#8242;,6-diamidino-2-phenylindole (DAPI, 0.2 &#181;g/mL) in 1x PBS solution.</li>
	<li>Incubate the samples for 10 min at RT in the dark.</li>
	<li>Aspirate the DAPI solution, and wash the cells twice with 1x PBS.</li>
	<li>Place two drops of 10 &#181;L each of mounting medium; ensure that the drops are separated sufficiently to allow two coverslips to be placed on a single slide.</li>
	<li>Remove excess liquid by tapping the coverslips on a clean towel, and then place them in an antifade mounting medium with the cells facing down.</li>
	<li>Place the mounted samples on a dry, flat surface in the dark and let them cure.</li>
	<li>Following curing, seal the edges of the coverslips with VALAP sealant or nail polish to prevent the samples from drying out.</li>
</ol>
2. SARS-CoV-2 RNA-FISH in HAE cultures
DAY 1
<ol>
	<li>Fixing and permeabilizing the HAE culture
	<ol>
		<li>Aspirate the medium, and fix the infected cells using 3.7% PFA solution for 1 h at RT.</li>
		<li>Aspirate the 3.7% PFA solution, and wash the cells twice with 1x PBS.
		<ol>
			<li>Replace the 3.7% PFA solution with 1x PBS under a Transwell insert.</li>
		</ol>
		</li>
		<li>Discard the PBS, and dehydrate the samples using 2 x 5 min washes with 100% MeOH prechilled to -20 &#176;C.</li>
		<li>After the second wash, replace the buffer with fresh, chilled MeOH for permeabilization under the Transwell insert. Store overnight at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
DAY 2
<ol start="2">
	<li>Rehydration 
	Rehydrate the samples through a graded series of MeOH/PBST solutions (each for 5 min) on ice: 75% MeOH/25% PBST, 50% MeOH/50% PBST, 25% MeOH/75% PBTS, and 100% PBST (twice).
	<ol>
		<li>Wash the cells for 5 min on ice with 50% 5x SSCT/50% PBST.</li>
		<li>Wash cells for 5 min on ice with 5x SSCT.</li>
		<li>Replace the 5x SSCT buffer with 1x PBS.</li>
	</ol>
	</li>
	<li>Detection
	<ol>
		<li>Incubate the cells (inside the Transwell insert) for 5 min on ice with 100 &#181;L of probe hybridization buffer. Next, transfer the plate to an incubator for 30 minutes at 37 &#176;C (prehybridization). 
		NOTE: The probe hybridization buffer must be pre-heated to 37 &#176;C before use. Calculate the required volume: 100 &#181;L is needed for a single Transwell insert.</li>
		<li>Prepare the probe solution. As 1 mL of probe solution requires 4 pmol of probe, add 4 &#181;L of 1 &#181;M probe stock solution to 1 mL of probe hybridization buffer, and mix well. 
		&#8203;NOTE: For RNA detection, use 100 &#181;L of probe solution per Transwell insert. Leave the probe solution on ice until the end of the prehybridization step.</li>
		<li>Remove the prehybridization solution, and add the probe solution.</li>
		<li>Incubate the cells overnight (12-18 h) at 37 &#176;C.&#8203;</li>
	</ol>
	</li>
</ol>
DAY 3
<ol start="5">
	<li>Remove excess probe by washing for 4 x 15 min with 100 &#181;L of probe wash buffer at 37 &#176;C.</li>
	<li>Wash the samples for 2 x 5 min with 5x SSCT at RT.</li>
	<li>Replace the 5x SSCT with 1x PBS, and store the samples at 4 &#176;C until amplification.</li>
</ol>
<ol start="4">
	<li>Amplification</li>
</ol>
<ol>
	<li>Preamplify the samples by incubating them with an amplification buffer for 30 min at RT.</li>
	<li>Prepare each hairpin by snap-cooling the desired volumes in separate tubes.
	<ol>
		<li>To prepare 500 &#181;L of amplification solution, use 30 pmol of each hairpin (e.g., for 500 &#181;L, use 10 &#181;L of 3 &#181;M stock hairpin solution).</li>
		<li>Transfer the hairpin solution to the tubes.</li>
		<li>Incubate at 95 &#176;C for 90 s.</li>
		<li>Cool to RT for 30 min in the dark.</li>
	</ol>
	</li>
	<li>Prepare the hairpin solution by adding all snap-cooled hairpins to 500 &#181;L of amplification buffer at RT.</li>
	<li>Remove the preamplification solution, and add the complete hairpin solution.</li>
	<li>Incubate the samples overnight (12-18 h) at RT in the dark.</li>
	<li>Remove excess hairpins by washing with 5x SSCT at RT as follows: 2 x 5 min, 2 x 15 min, and 1 x 5 min.</li>
	<li>Replace the 5x SSCT solution with 1x PBS, and store at 4 &#176;C for not more than 2-3 days, or proceed directly to nuclear staining. 
	NOTE: If required, use the RNA-FISH samples in a standard immunofluorescence assay, followed by nuclear staining (see section 3).</li>
</ol>
5. Nuclear staining and slide mounting
<ol>
	<li>Aspirate the 1x PBS solution, and replace it with DAPI solution (0.2 &#181;g/mL) in 1x PBS.</li>
	<li>Incubate the samples for 10 min at RT in the dark.</li>
	<li>Aspirate the DAPI solution, and wash the cells twice with 1x PBS.</li>
	<li>Place the cut-out membrane from the Transwell inserts onto 10 &#181;L of antifade mounting medium with the cells facing up, and add extra mounting medium (5 &#181;L) to the membrane.</li>
	<li>Cover the membranes with coverslips.</li>
	<li>Cure the mounted samples on a dry, flat surface in the dark.</li>
	<li>Following curing, seal the edges of the coverslips with VALAP sealant or nail polish to prevent the samples from drying out.</li>
</ol>
3. Immunofluorescence analysis of Vero cells and HAE cultures
NOTE: Perform the immunofluorescence assay on day 3 for cell lines or day 4 for HAE cultures. Use a different approach for each model. All differences are indicated clearly.
<ol>
	<li>Aspirate the 1x PBS solution from the wells.</li>
	<li>Block the samples by incubation with 1% w/v bovine serum albumin in PBST solution for 30 min at 37 &#176;C.</li>
	<li>Prepare primary antibodies by preparing appropriate dilutions in blocking solution, and incubate.
	<ol>
		<li>For Vero cells on coverslips:
		<ol>
			<li>Place drops (30-50 &#181;L) of antibody solution onto parafilm in a humidity chamber.</li>
			<li>Place coverslips onto the antibody drops with the cells facing down.</li>
		</ol>
		</li>
		<li>For HAE, replace the blocking agent in the inserts with antibody solution (100 &#181;L), and incubate the samples in a humidified chamber. 
		NOTE: Adjust the time and temperature for each incubation with the primary antibody. Typical parameters are 2 h at RT or overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Wash the samples for 3 x 5 min with PBST.
	<ol>
		<li>For cells on coverslips, transfer to a 12-well plate and add PBST solution.</li>
		<li>For HAE cultures, replace the antibody solution with the PBST solution.</li>
	</ol>
	</li>
	<li>Check the light sources and filters available for the confocal microscope.</li>
	<li>Choose secondary antibodies according to the host species. Choose the fluorophore parameters (excitation and emission wavelengths, spectrum width, and excitation efficiency according to the available light source). 
	NOTE: Spectral parameters can be modeled using online tools (see Table of Materials).</li>
	<li>Prepare appropriate secondary antibody solutions by diluting them with a blocking solution.</li>
	<li>Incubate with secondary antibodies (as in 6.3), but incubate for 1 h at 37 &#176;C.</li>
	<li>Wash the samples as in step 3.4.</li>
	<li>Nuclear staining and slide mounting
	<ol>
		<li>For cells on coverslips
		<ol>
			<li>Aspirate the PBST, and replace it with DAPI (0.2 &#181;g/mL) in 1x PBS solution.</li>
			<li>Incubate the samples for 10 min at RT in the dark.</li>
			<li>Aspirate the DAPI solution, and wash the cells twice with 1x PBS.</li>
			<li>Place the coverslips onto drops of 10 &#181;L of mounting medium with the cells facing down.</li>
			<li>Seal the coverslips with nail polish.</li>
		</ol>
		</li>
		<li>For HAE cultures
		<ol>
			<li>Aspirate the 1x PBST, and replace it with DAPI (0.2 &#181;g/mL) in 1x PBS solution.</li>
			<li>Incubate the samples for 10 min at RT in the dark.</li>
			<li>Aspirate the DAPI solution, and wash the cells twice with 1x PBS.</li>
			<li>Place the cut-out membranes onto drops of 10 &#181;L of mounting medium with the cells facing up, and add an extra (5 &#181;L) mounting medium to the membrane.</li>
			<li>Cover the membranes with coverslips.</li>
			<li>Seal the coverslips with nail polish.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Confocal microscopy
<ol>
	<li>Define the tracks by specifying the fluorophores used.</li>
	<li>Choose the scanning mode and speed.</li>
	<li>Adjust the laser power, gain, and offset values for each fluorophore by comparing them with respective negative controls: for virus, mock-infected cells; for cellular proteins, samples stained with isotype control antibodies from an appropriate host.</li>
	<li>To acquire a 3D image, activate z-stack mode, and set the top and bottom limits. Set the step size/number. 
	NOTE: For more details on coronavirus imaging.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope LSM 880</td>
			<td>ZEISS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator Galaxy170R</td>
			<td>New Brunswick</td>
			<td>CO170R-230-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mm x 15 mm NO. 1 coverslips</td>
			<td>LabSolute</td>
			<td>7695022</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>FL-MEDICAL</td>
			<td>5.350.023.053</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>TTP</td>
			<td>92412</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluorophore 488-conjugated secondary antibodies</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;5-tubulin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-134234</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>HCR Amplification Buffer</td>
			<td>Molecular Instruments, Inc</td>
			<td>BAM01522</td>
			<td>Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td>
		</tr>
		<tr>
			<td>HCR amplifier B1-h1 Alexa Fluor 647</td>
			<td>Molecular Instruments, Inc.</td>
			<td>S013922</td>
			<td></td>
		</tr>
		<tr>
			<td>HCR amplifier B1-h2 Alexa Fluor 647</td>
			<td>Molecular Instruments, Inc.</td>
			<td>S012522</td>
			<td></td>
		</tr>
		<tr>
			<td>HCR Probe Hybridization Buffer</td>
			<td>Molecular Instruments, Inc.</td>
			<td>BPH03821</td>
			<td>Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td>
		</tr>
		<tr>
			<td>HCR probe set for SARS-CoV-2 Ncapsid</td>
			<td>Molecular Instruments, Inc.</td>
			<td>PRE134</td>
			<td></td>
		</tr>
		<tr>
			<td>HCR Probe Wash Buffer</td>
			<td>Molecular Instruments, Inc.</td>
			<td>BPW01522</td>
			<td>Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td>
		</tr>
		<tr>
			<td>Fluorescence Spectraviewer</td>
			<td></td>
			<td></td>
			<td>Modeling spectral parameters</td>
		</tr>
		<tr>
			<td>ImageJ Fiji</td>
			<td></td>
			<td></td>
			<td>Acquiring and processing z-stack images</td>
		</tr>
		<tr>
			<td>ImmunoFluorescence (IF)</td>
			<td>Blocking</td>
			<td>(1% BSA in PBST) 30 min at 37 &#176;C</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Primary antibody incubation</td>
			<td>(Antibody solution of apropriate concentration in blocking solution) 2 h at room temperature / overnight at 4 &#176;C, 30-50 &#181;L</td>
			<td>(Antibody solution of apropriate concentration in blocking solution) 2 h at room temperature / overnight at 4 &#176;C, 100 &#181;L</td>
		</tr>
		<tr>
			<td></td>
			<td>Primary antibody washing</td>
			<td>(PBST) 3 x 5 min at room temperature</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Secondary antibody incubation</td>
			<td>(Antibody solution of apropriate concentration in blocking solution) 1 h at 37 &#176;C, 30-50 &#181;L</td>
			<td>(Antibody solution of appropriate concentration in blocking solution) 1 h at 37 &#176;C, 100 &#181;L</td>
		</tr>
		<tr>
			<td></td>
			<td>Secondary antibody washing</td>
			<td>(PBST) 3 x 5 min at room temperature</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Nuclear staining</td>
			<td>(DAPI solution) 10 min at room temperature, then 2 x 5 min with 1x PBS</td>
			<td></td>
		</tr>
	</tbody>
</table>

using immuno-rna fluorescence in situ hybridization to visualize sars-cov-2

Source: Kula-Pacurar, A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/62067">Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization.</a> J. Vis. Exp. (166), (2020).
This video demonstrates the application of Hybridization Chain Reaction (HCR)-Fluorescence In Situ Hybridization (FISH) for visualizing the spatial distribution of SARS-CoV-2 subgenomic RNA. Combining HCR with immunofluorescence techniques has the potential to provide insights into host-virus interactions at the single-cell level.

Using Immuno-RNA Fluorescence In Situ Hybridization to Visualize SARS-CoV-2

1. SARS-CoV-2 RNA-FISH in Vero cells cultured on coverslips
DAY 1 

	Fixing and permeabilizing cells&#8203;
	
		Fix infected cells with 3.7% w/v PFA ...

Take fixed and permeabilized coronavirus-infected Vero cells &#8212; an interferon-deficient mammalian cell line.
Inside the host, viral RNA is present as subgenomic or sgRNAs, intermediate-length RNA molecules encoding specific viral proteins synthesized via transcription.
Add hybridization chain reaction or HCR initiator probes.
The HCR probe binds to its complementary sequence on the target RNA.
Introduce fluorescently labeled oligonucleotide hairpin probes, H-1 and H-2.
The initiator probe binds to its complementary H1 tail, linearizing the hairpin structure.
The exposed H1 sequence binds to its complementary H2 tail.
The trailing H2 sequence continues binding to an H1 tail, amplifying fluorescent-tagged probes around the target area, and generating a robust signal.
Add appropriate antibodies to visualize the host cell cytoskeleton.
Stain the nuclei using DAPI.
Under a fluorescence microscope, virus-infected cells display red fluorescence in the cytoplasm, indicating the presence of the target RNA.

using-immuno-rna-fluorescence-situ-hybridization-to-visualize-sars

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

179 Views. Źródło: Kula-Pacurar, A., et al. Wizualizacja SARS-CoV-2 za pomocą hybrydyzacji immuno-fluorescencji in situ. J. Vis. Exp. (166), (2020). Ten film demonstruje zastosowanie hybrydyzacji reakcji łańcuchowej (HCR)-fluorescencji in situ (FISH) do wizualizacji przestrzennego rozkładu subgenomowego RNA SARS-CoV-2. Połączenie HCR z technikami immunofluorescencji może dostarczyć informacji na temat interakcji gospodarz-wirus na poziomie pojedynczej komórki. 

Video: Wykorzystanie fluorescencji immuno-RNA Hybrydyzacja in Situ do wizualizacji SARS-CoV-2 - Concept

Transcriptional Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells differentiate into primary trophoblast giant cells (TGCs) while the polar trophectoderm overlying the inner cell mass continues to proliferate later differentiating into secondary TGCs. TGCs play a key role in developing placenta and are essential for a successful pregnancy. Investigation of transcriptional regulation of specific genes during post-implantation development can give insights into TGCs development. Cells of the ectoplacental cone (EPC) from embryos at 7-7.5 days of gestation (E7-7.5), derived from the polar trophectoderm, differentiate into secondary TGCs1. TGCs can be studied in situ, on cryostat sections of embryos at E7 although the number of TGCs is very low at this stage. An alternative means of analyzing secondary TGCs is to use short-term cultures of individual EPCs from E7 embryos. We propose a technique to investigate the transcriptional status of genes of interest both in vivo and in vitro at the single-cell level using fluorescent in situ hybridization (RNA FISH) to visualize nascent transcripts. This technique provides a direct readout of gene expression and enables assessment of the chromosomal status of TGCs, which are large endoreplicating cells. Indeed, a key feature of terminal differentiation of TGCs is that they exit the cell cycle and undergo multiple rounds of endoreplication.This approach can be applied to detect expression of any gene expressed from autosomes and/or sex chromosomes and can provide important information into developmental mechanisms as well as placental diseases.

Transcriptional  Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

Trophoblast giant cells (TGCs) play a key role in the placenta to ensure a healthy pregnancy. We present a protocol for assessing the transcriptional status of genes in TGCs by nascent fluorescent in situ hybridization on cryostat sections of post-implantation embryos or short-term cultures of embryonic day 7 ectoplacental cones.

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells ...

JoVE Journal

Developmental Biology

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

Immunology and Infection

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

Using Immuno-RNA Fluorescence In Situ Hybridization to Visualize SARS-CoV-2

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Using Immuno-RNA Fluorescence In Situ Hybridization to Visualize SARS-CoV-2