The authors would like to thank Carolina Tropini, Katharine Ng, Justin Sonnenburg, and KC Huang for guidance in developing the FISH technique. Teresa O&rsquo;Meara provided helpful comments on the manuscript, and Miriam Levy assisted with photography. This work was supported by NIH grants R01AI108992, R01DK113788, and a Burroughs Welcome Award in the Pathogenesis of Infectious Disease.

<ol>
	<li>Noble, S. M., Gianetti, B. A., Witchley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+Albicans+Cell-Type+Switching+and+Functional+Plasticity+in+the+Mammalian+Host.">Candida Albicans Cell-Type Switching and Functional Plasticity in the Mammalian Host.</a> Nature Reviews in Microbiology. 15 (2), 96-108 (2017).</li><li>Bacher, 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=Human+Anti-fungal+Th17+Immunity+and+Pathology+Rely+on+Cross-Reactivity+against+Candida+albicans.">Human Anti-fungal Th17 Immunity and Pathology Rely on Cross-Reactivity against Candida albicans.</a> Cell. 176 (6), 1340-1355 (2019).</li><li>Shao, T. Y., 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=Commensal+Candida+albicans+Positively+Calibrates+Systemic+Th17+Immunological+Responses.">Commensal Candida albicans Positively Calibrates Systemic Th17 Immunological Responses.</a> Cell Host and Microbe. 25 (3), 404-417 (2019).</li><li>Jiang, T. 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=Commensal+Fungi+Recapitulate+the+Protective+Benefits+of+Intestinal+Bacteria.">Commensal Fungi Recapitulate the Protective Benefits of Intestinal Bacteria.</a> Cell Host and Microbe. 22 (6), 809-816 (2017).</li><li>Iliev, I. D., 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=Interactions+Between+Commensal+Fungi+and+the+C-Type+Lectin+Receptor+Dectin-1+Influence+Colitis.">Interactions Between Commensal Fungi and the C-Type Lectin Receptor Dectin-1 Influence Colitis.</a> Science. 336 (6086), 1314-1317 (2012).</li><li>Fan, D., 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=Activation+of+HIF-1+alpha+and+LL-37+by+Commensal+Bacteria+Inhibits+Candida+Albicans+Colonization.">Activation of HIF-1 alpha and LL-37 by Commensal Bacteria Inhibits Candida Albicans Colonization.</a> Nature Medicine. 21 (7), 808-814 (2015).</li><li>Koh, A. Y., Kohler, J. R., Coggshall, K. T., Van Rooijen, N., Pier, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+Damage+and+Neutropenia+Are+Required+for+Candida+Albicans+Dissemination.">Mucosal Damage and Neutropenia Are Required for Candida Albicans Dissemination.</a> PLoS Pathogens. 4 (2), e35 (2008).</li><li>Yamaguchi, N., 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=Gastric+Colonization+of+Candida+Albicans+Differs+in+Mice+Fed+Commercial+and+Purified+Diets.">Gastric Colonization of Candida Albicans Differs in Mice Fed Commercial and Purified Diets.</a> Journal of Nutrition. 135 (1), 109-115 (2005).</li><li>Kadosh, D., 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=Effect+of+Antifungal+Treatment+in+a+Diet-Based+Murine+Model+of+Disseminated+Candidiasis+Acquired+via+the+Gastrointestinal+Tract.">Effect of Antifungal Treatment in a Diet-Based Murine Model of Disseminated Candidiasis Acquired via the Gastrointestinal Tract.</a> Antimicrobial Agents and Chemotherapy. 60 (11), 6703-6708 (2016).</li><li>Perlroth, J., Choi, B., Spellberg, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+Fungal+Infections:+Epidemiology,+Diagnosis,+and+Treatment.">Nosocomial Fungal Infections: Epidemiology, Diagnosis, and Treatment.</a> Medical Mycology. 45 (4), 321-346 (2007).</li><li>Pande, K., Chen, C., Noble, S. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+Albicans+Cell-Type+Switching+and+Functional+Plasticity+in+the+Mammalian+Host.">Candida Albicans Cell-Type Switching and Functional Plasticity in the Mammalian Host.</a> Nature Reviews in Microbiology. 15 (2), 96-108 (2017).</li><li>Balish, E., Filutowicz, H., Oberley, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlates+of+Cell-Mediated+Immunity+in+Candida+Albicans-Colonized+Gnotobiotic+Mice.">Correlates of Cell-Mediated Immunity in Candida Albicans-Colonized Gnotobiotic Mice.</a> Infection and Immunity. 58 (1), 107-113 (1990).</li><li>Guarner, J., Brandt, M. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Amino+Acid+Liquid+Synthetic+Medium+for+the+Development+of+Mycelial+and+Yeast+Forms+of+Candida+Albicans.">An Amino Acid Liquid Synthetic Medium for the Development of Mycelial and Yeast Forms of Candida Albicans.</a> Sabouraudia. 13 (2), 148-153 (1975).</li><li>Banerjee, M., 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=UME6,+a+Novel+Filament-Specific+Regulator+of+Candida+Albicans+Hyphal+Extension+and+Virulence.">UME6, a Novel Filament-Specific Regulator of Candida Albicans Hyphal Extension and Virulence.</a> Molecular Biology of the Cell. 19 (4), 1354-1365 (2008).</li><li>Earle, K. 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=Quantitative+Imaging+of+Gut+Microbiota+Spatial+Organization.">Quantitative Imaging of Gut Microbiota Spatial Organization.</a> Cell Host and Microbe. 18 (4), 478-488 (2015).</li><li>Sokol, H., 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=Fungal+Microbiota+Dysbiosis+in+IBD.">Fungal Microbiota Dysbiosis in IBD.</a> Gut. 66 (6), 1039-1048 (2017).</li></ol>

The authors have nothing to disclose.

The method described here allows for visualization of C. albicans yeasts and hyphae in the GI tracts of commensally colonized mice of any sex or strain. The FISH probe hybridizes to 23S rRNA, which is distributed throughout the fungal cytoplasm. Our method was adapted from a previously reported protocol for visualizing gut bacteria20. Because C. albicans changes its morphology within the host, the method is useful to monitor fungal cell shape as well as localization. For example, we have used this method to disprove the hypothesis that yeasts predominate throughout the digestive tract, and to expose discrepancies between the in vivo and in vitro phenotypes of certain &ldquo;filamentation-defective&rdquo; mutants12.
Several mouse models exist for C. albicans commensal colonization. The microbiota of laboratory mice and humans are distinct and, in mice, the use of antibiotics or a specialized diet is required to establish stable colonization. Treatment with antibiotics also enhances C. albicans colonization of humans and is a major risk factor for disseminated disease10. The antibiotics used in this study are relatively inexpensive and yield reliable decreases in the bacterial microbiota. Note that antibiotics are used to decrease the burden of antagonistic bacterial species, not to eliminate all bacteria from the animals. If researchers wish to study C. albicans-host interactions in the absence of bacteria or to avoid the use of antibiotics or a special diet, germ-free animals can be substituted for conventionally reared animals; however, gnotobiotic mice exhibit certain immune and anatomic abnormalities and therefore may not be suitable for all purposes.
Several steps are important for successful FISH staining: The recommended fixation method is critical to preserve the structural integrity of GI tissues, particularly the fragile contents of the gut lumen, such as the mucus layer and the three-dimensional organization of fungi and bacteria16. Please note that many commonly used fixation solutions that contain water are highly damaging to the luminal architecture. The fixatives and solutions for post-fixation washes recommended in this protocol do not contain water, and it is important to avoid contamination with water. Another critical step is the hybridization step, where it is important to avoid evaporation of the hybridization solution during incubation of slides in the hybridization oven. We suggest placing the slides in a watertight container to avoid this problem. Alternatively, one can use watertight hybridization chambers such as those originally used for hybridization of microarrays.
A C. albicans-specific FISH probe is described in this protocol. However, because many laboratory-reared mice do not contain C. albicans or other fungi as part of their natural gut microbiota, a panfungal probe may specifically stain C. albicans in experimentally colonized animals. Substitution of a panfungal probe may be desirable because of its superior hybridization characteristics and higher signal-to-noise ratio. If the panfungal probe is used as a pseudospecific probe for C. albicans, it is important to document a lack of staining in uninfected animals (i.e., treated with antibiotics but not C. albicans). Staining of a noncolonized control animal is also useful to assess background staining that may result from adherence of FISH probes to food particulates. Overall, the use of FISH probes offers enhanced specificity over most other methods of staining fungi, such as Calcofluor white (which stains chitin, a cell wall component that may vary between cell types), GMS, or commercially available antifungal antibodies. Moreover, FISH staining allows for costaining of multiple organisms using differentially labeled FISH probes to species-specific rRNAs.
One caveat of this technique is that certain C. albicans yeast cell types appear very similar by FISH (for example, the opaque and GUT cell types). To distinguish among these cell types, it would be useful to develop hybridization probes to cell type-specific fungal mRNAs. Nevertheless, in its current form, the FISH technique has already revealed surprising discrepancies between the in vivo and in vitro behaviors of C. albicans mutants evaluated under both conditions12, indicating complex interactions in the natural commensal environment that are not adequately mimicked by existing in vitro assays. Further studies of different C. albicans stains in additional wild type and mutant hosts are likely to yield additional insights into fungal-host interactions. In particular, it will be instructive to profile C. albicans in models of inflammatory bowel disease, which is associated with high titers of C. albicans in humans21, and other models of C. albicans overgrowth and disease. The FISH technique may also be combined with immunohistochemistry to stain specific host cells. Overall, the method presented here provides a reasonably quick and reliable means to determine C. albicans localization and morphology within the mammalian GI tract.

Candida albicans is a fungal commensal as well as an opportunistic human pathogen. This yeast lacks a defined environmental niche and instead propagates within the gastrointestinal (GI) tract, skin, and genitourinary tract of humans and other mammals1. Whereas early research on C. albicans focused primarily on its virulence potential, several recent reports suggest that commensally propagating organisms within the gut may play important roles in normal health, including the immune development of the host2,3,4. To facilitate investigations of C. albicans commensalism within the mammalian gut, we developed a mouse model of stable GI colonization and a fluorescence in situ hybridization (FISH)-based method to visualize fungal yeast cells and hyphae within the intestinal lumen.
With some exceptions5, laboratory-bred mice typically exhibit resistance to fungal colonization of the digestive tract. Colonization resistance is thought to be mediated by specific bacterial species; however, this can be overcome by treatment of the animals with antibiotics6,7 or use of a chemically-defined diet that presumably alters bacterial species composition8,9. Similarly, in humans, the use of broad-spectrum antibiotics has been associated with C. albicans overgrowth and dissemination10. Our murine colonization model uses broad-spectrum antibiotics to establish C. albicans colonization of immunocompetent, conventionally reared mice. Penicillin and streptomycin are provided in the animals&#8217; drinking water for one week prior to gavage with 108 colony forming units (CFUs) of C. albicans. As long as the antibiotic-infused water is continued, C. albicans will propagate through the GI tract, reaching fecal titers of 106&#8722;108 CFUs/g. Despite the high level of fungal colonization, animals remain healthy and gain weight at the same rate as uninfected controls. This model has been used successfully to screen for and characterize multiple C. albicans commensalism factors11,12.
Like other members of the fungal kingdom, C. albicans is capable of enormous morphological plasticity13. Under in vitro conditions, it has been shown to transition among at least six unicellular yeast cell types, as well as multicellular hyphae and pseudohyphae. The yeast-to-hypha transition is one of its best-characterized virulence attributes, and hyphae and pseudohyphae predominate in most mammalian disease models, as well as in infected human tissues. To determine C. albicans localization and cell morphology within the murine digestive tract, we developed a FISH technique for staining yeasts and hyphae in fixed histological sections. The probes consist of fluorescently labeled DNA oligonucleotides that hybridize to fungal 23S ribosomal RNA (rRNA), which is distributed throughout the fungal cytoplasm. Because host tissues are fixed in a manner that preserves the three-dimensional architecture of the gut, including the host mucosa, digestive material, the bacterial microbiota, and mucus within the gut lumen, this technique permits the localization of fungal cells with respect to these landmarks when stained. The FISH technique compares favorably to traditional histological stains for fungi, such as Periodic Acid Schiff (PAS) or Gomori&#39;s Methenamine-Silver (GMS), as well as commercially available antifungal antibodies, because these reagents are not specific for C. albicans. Moreover, standard fixatives remove the mucus layer and disrupt other contents of the gut lumen14,15.
In this article, we provide detailed instructions for establishing high-grade C. albicans colonization of the mouse GI tract, for dissection of the digestive tract from euthanized animals, for tissue fixation in a manner that preserves luminal architecture, and for detection of C. albicans within host tissues using FISH. In addition to wild type and mutant strains of C. albicans, the gavage technique can be used to deliver other microorganisms. The fixation technique would be useful for any study in which preservation of gut contents is desired. The FISH procedure can be completed within a day and can be used to localize multiple fungal species by using multiple, differentially labeled probes.

<table>
	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td>can be substituted from any vendor</td>
		</tr>
		<tr>
			<td>BHI blood agar</td>
			<td></td>
			<td></td>
			<td>can be substituted from any vendor</td>
		</tr>
		<tr>
			<td>C. albicans FISH probe</td>
			<td>IDT DNA Technologies</td>
			<td>custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>chamber for hybridization incubation</td>
			<td></td>
			<td></td>
			<td>watertight chamber meant to reduce evaporation</td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>Sigma</td>
			<td>C2432</td>
			<td>&#62;=99.5%</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Roche</td>
			<td>10236276001</td>
			<td>can be substituted from any vendor</td>
		</tr>
		<tr>
			<td>delicate task wiper tissues</td>
			<td>Kimberley-Clark</td>
			<td>34256CT</td>
			<td>Kimwipes</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma</td>
			<td>G7021-5KG</td>
			<td>can be substituted from any vendor</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Sigma</td>
			<td>E7023</td>
			<td>molecular biology grade</td>
		</tr>
		<tr>
			<td>feeding needles</td>
			<td>Cadence, Inc.</td>
			<td>7910</td>
			<td>metal; 20 X1-1/2&#34; W/2-1/4; can be autoclaved to sterilize</td>
		</tr>
		<tr>
			<td>FITC-UEA-1</td>
			<td>Sigma</td>
			<td>L9006-1MG</td>
			<td>other fluorophores available</td>
		</tr>
		<tr>
			<td>FITC-WGA</td>
			<td>Sigma</td>
			<td>L4895-2MG</td>
			<td>other fluorophores available</td>
		</tr>
		<tr>
			<td>Foam pads</td>
			<td>Fisher</td>
			<td>22038221</td>
			<td>Order foam pads that will fit within cassettes</td>
		</tr>
		<tr>
			<td>formamide</td>
			<td>Sigma</td>
			<td>47671</td>
			<td>molecular biology grade</td>
		</tr>
		<tr>
			<td>glacial acetic acid</td>
			<td>Macron Fine Chemicals</td>
			<td>MK881746</td>
			<td>ACS reagent, &#62;=99.5%</td>
		</tr>
		<tr>
			<td>glass Coplin jar</td>
			<td>Fisher</td>
			<td>08-815</td>
			<td>hold up to 10 slides back to back</td>
		</tr>
		<tr>
			<td>Histology cassettes</td>
			<td>Simport</td>
			<td>M512</td>
			<td>Deep cassettes so cecum is not squished</td>
		</tr>
		<tr>
			<td>hybridization coverslips</td>
			<td>Sigma</td>
			<td>GBL712222</td>
			<td>RNase-free</td>
		</tr>
		<tr>
			<td>hybridization oven</td>
			<td></td>
			<td></td>
			<td>can be substituted from any vendor</td>
		</tr>
		<tr>
			<td>LB</td>
			<td></td>
			<td></td>
			<td>can be substituted from any vendor</td>
		</tr>
		<tr>
			<td>Lee&#39;s media</td>
			<td></td>
			<td></td>
			<td>prepared as described in Lee et al. 1975</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sigma</td>
			<td>179337</td>
			<td>ACS reagent, &#62;=99.8%</td>
		</tr>
		<tr>
			<td>mice</td>
			<td>Charles River Laboratories</td>
			<td>028</td>
			<td>adult BALB/c; 18-21 grams (8-10 weeks)</td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Fisher</td>
			<td>50-980-487</td>
			<td>16% solution</td>
		</tr>
		<tr>
			<td>parrafin wax</td>
			<td>Sigma</td>
			<td>P3558-1KG</td>
			<td>Paraplast for tissue embedding</td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>UCSF Cell Culture Facility Media Production Unit</td>
			<td>CCFAL003</td>
			<td>calcium, magnesium-free; can be substituted from any vendor</td>
		</tr>
		<tr>
			<td>penicillin G</td>
			<td>Sigma</td>
			<td>PENNA-100MU</td>
			<td></td>
		</tr>
		<tr>
			<td>Sabouraud dextrose agar</td>
			<td></td>
			<td></td>
			<td>can be substituted from any vendor</td>
		</tr>
		<tr>
			<td>saline</td>
			<td>Baxter</td>
			<td>2F7123</td>
			<td>sterile, can be substituted from any vendor</td>
		</tr>
		<tr>
			<td>sodium chloride (NaCl)</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td>can be substituted from any vendor; maintain RNase-free</td>
		</tr>
		<tr>
			<td>sodium dodecyl sulfate (SDS)</td>
			<td>Sigma</td>
			<td>L3771</td>
			<td>can be substituted from any vendor; maintain RNase-free</td>
		</tr>
		<tr>
			<td>streptomycin</td>
			<td>Sigma</td>
			<td>S9137-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>super PAP pen</td>
			<td>Life Technologies</td>
			<td>8899</td>
			<td>can be substituted from any vendor</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma</td>
			<td>T3253</td>
			<td>can be substituted from any vendor; maintain RNase-free</td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>does not contain DAPI</td>
		</tr>
		<tr>
			<td>xylenes</td>
			<td>Sigma</td>
			<td>214736</td>
			<td>reagent grade</td>
		</tr>
		<tr>
			<td>YEPD</td>
			<td></td>
			<td></td>
			<td>can be substituted from any vendor</td>
		</tr>
	</tbody>
</table>

visualization of candida albicans in the murine gastrointestinal tract using fluorescent in situ hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Following the instructions provided, the outcome of this technique will be fluorescent labelling of nuclei in the host epithelium, mucus, and C. albicans in sectioned GI tissues. Wild type Candida albicans cells should appear as either round, unicellular yeast cells or highly elongated, sometimes branching, multicellular hyphae (cell-cell divisions will not be apparent in the hyphae). Mutants of C. albicans may additionally appear as smaller, slightly elongated &#8220;gray&#8221; yeasts or as larger, slightly elongated &#8220;GUT&#8221; (gastrointestinal-induced transition) or &#8220;opaque&#8221; yeasts.
Figure 1 depicts the feeding needle used for oral gavage, as well as key positions of the needle during the gavage procedure. Figure 2 provides a typical setup used for organ dissection and the appearance of GI segments of the mouse.
Figure 3 displays FISH staining of different C. albicans cell types following propagation in vitro and within the mouse model. Figure 3A shows fluorescence and phase images of fixed, permeabilized, in vitro-propagated hyphae. Hypha formation was induced with liquid Lee&#8217;s medium18 with 2% N-acetylglucosamine, pH 6.8 at 37 &#176;C. Figure 3B shows fluorescence and phase images of fixed, permeabilized, in vitro-propagated yeast cells. Figure 3C,D shows fixed, permeabilized, in vitro-propagated GUT cells and opaque cells, respectively. GUT and opaque cell types are morphologically indistinguishable except when visualized by scanning electron microscopy. Please note that FISH staining of in vitro-propagated cells will be suboptimal if the cells are not well permeabilized. An example of weak and diffuse staining is shown in Figure 3C. For best results, cell fixation and permeabilization conditions should be determined empirically for each cell type and species to be investigated. Fortunately, the recommended protocol for visualizing C. albicans in sectioned tissues produces more consistent permeabilization.
Figure 3E-G depict C. albicans in mouse large intestines, stained with the C. albicans-specific probe (Figure 3E) or the panfungal probe (Figure 3F,G). C. albicans appears red in these images, host cell nuclei are blue, and the mucus layer is green. Both round yeasts and highly elongated hyphae (white arrowheads) occur in the large intestines. Please note that staining is brighter with the panfungal probe. Figure 3G depicts a ume6 mutant in the same compartment, stained with the panfungal probe. Ume6 encodes a transcription factor that is required for hypha formation under in vitro conditions19. Interestingly, the yeast-locked phenotype displayed by this mutant under in vitro conditions is not recapitulated within the gut, suggesting that a redundant factor must be activated within the host12. Figure 4 depicts the appearance of wild type C. albicans in different segments of the murine GI tract, including the regions immediately adjacent to the host mucosa and more central regions of the gut lumen.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60283/60283fig01.jpg" /> 
Figure 1: Key feeding needle positions during gavage. (A) Feeding needle. (B) Feeding needle ball inserted to the side of the tongue. (C) Feeding needle in the upright position with insertion in the esophagus. (D) Feeding needle inserted into the stomach with a few millimeters visible above the nose. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60283/60283fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60283/60283fig02.jpg" /> 
Figure 2: Example dissection layout. (A) Dissection pad and tools. (B) Excised GI tract. Proximal (esophagus) to distal (anus) sections: I. Stomach. II. Proximal small intestines. III. Medial small intestines. IV. Distal small intestines. V. Cecum. VI. Large intestine. Pink dashed border boxes indicate the tissues to be fixed. (C) Tissues positioned in cassettes. Roman numerals are the same as in panel B. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60283/60283fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60283/60283fig03.jpg" /> 
Figure 3: Representative images of FISH-stained C. albicans. (A) FISH of C. albicans hyphae grown in liquid Lee&#8217;s media18 with 2% N-acetylglucosamine, pH 6.8. (B) FISH of C. albicans round yeast cells grown on YEPD + 2% agar plates. (C) FISH of C. albicans GUT cells (ySN1045) grown on YEPD + 2% agar plates. (D) FISH of C. albicans opaque cells grown on YEPD + 2% agar plates. (E) Wild type C. albicans (ySN250) in large intestines, stained with the C. albicans-specific probe. (F) Wild type C. albicans in large intestines, stained with the panfungal probe. (G) Ume6 mutant (ySN1479) in large intestines, stained with the panfungal probe: red is C. albicans, blue is host epithelial nuclei, and green is mucin. Arrowheads indicate hyphae. Arrows indicate yeast. Scale bars = 20 &#181;m. Images F and G are adapted from Witchley et al.12. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60283/60283fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60283/60283fig04.jpg" /> 
Figure 4: Appearance of FISH-stained C. albicans in different gut compartments. Wild type C. albicans is shown in the indicated segments of the mouse GI tract. Within each compartment, images depict the area adjacent to the host mucosa and the central region of the gut lumen. Staining was performed with the panfungal probe.&#160;Scale bar = 20 &#181;m. Images are adapted from Witchley et al.12. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60283/60283fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Antibiotic stocks (200x)</td>
		</tr>
		<tr>
			<td>Penicillin G</td>
			<td>181 mg/mL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>400 mg/&#181;L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Methacarn</td>
		</tr>
		<tr>
			<td>Stock</td>
			<td>Final concentration</td>
			<td>Volume</td>
			<td>Units</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>60%</td>
			<td>300</td>
			<td>mL</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>30%</td>
			<td>150</td>
			<td>mL</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>10%</td>
			<td>50</td>
			<td>mL</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Hybridization solution</td>
		</tr>
		<tr>
			<td>Stock</td>
			<td>Final concentration</td>
			<td>Volume</td>
			<td>Units</td>
		</tr>
		<tr>
			<td>1 M Tris-HCl, pH 7.4</td>
			<td>20 mM</td>
			<td>20</td>
			<td>&#181;L</td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>0.9 M</td>
			<td>180</td>
			<td>&#181;L</td>
		</tr>
		<tr>
			<td>10% SDS</td>
			<td>0.10%</td>
			<td>10</td>
			<td>&#181;L</td>
		</tr>
		<tr>
			<td>100% formamide</td>
			<td>1.00%</td>
			<td>10</td>
			<td>&#181;L (kept in small aliquots at -20 &#176;C between uses)</td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td></td>
			<td>780</td>
			<td>&#181;L</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">FISH washing solution</td>
		</tr>
		<tr>
			<td>1 M Tris-HCl, pH 7.4</td>
			<td>20 mM</td>
			<td>1</td>
			<td>mL</td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>0.9 M</td>
			<td>9</td>
			<td>mL</td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td></td>
			<td>40</td>
			<td>mL</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Mucin-nuclei staining solution</td>
		</tr>
		<tr>
			<td>DAPI, 10 &#181;g/mL</td>
			<td>20 ng/mL</td>
			<td>2</td>
			<td>&#181;L</td>
		</tr>
		<tr>
			<td>lectin, 40 &#181;g/mL</td>
			<td>1.6 &#181;g/mL</td>
			<td>40</td>
			<td>&#181;L</td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td></td>
			<td>958</td>
			<td>&#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: Typical volumes and concentrations of solutions used in this protocol.

Watch this Scientific Journal Video about Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization at JoVE.com

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

visualization-candida-albicans-murine-gastrointestinal-tract-using

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

11.8K Views.  University of California, San Francisco. The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Video: Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...