Name: visualization of candida albicans in the murine gastrointestinal tract using fluorescent in situ hybridization
Uploaded: 2019-11-05T15:00:00
Duration: 10 min 8 s
Description: Watch this Scientific Journal Video about Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization at JoVE.com

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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passage+Through+the+Mammalian+Gut+Triggers+a+Phenotypic+Switch+That+Promotes+Candida+Albicans+Commensalism.">Passage Through the Mammalian Gut Triggers a Phenotypic Switch That Promotes Candida Albicans Commensalism.</a> Nature Genetics. 45 (9), 1088-1091 (2013).</li><li>Witchley, J. 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=Candida+Albicans+Morphogenesis+Programs+Control+the+Balance+between+Gut+Commensalism+and+Invasive+Infection.">Candida Albicans Morphogenesis Programs Control the Balance between Gut Commensalism and Invasive Infection.</a> Cell Host and Microbe. 25 (3), 432-443 (2019).</li><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>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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathologic+Diagnosis+of+Fungal+Infections+in+the+21st+Century.">Histopathologic Diagnosis of Fungal Infections in the 21st Century.</a> Clinical Microbiology Reviews. 24 (2), (2011).</li><li>Johansson, M. E., Hansson, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+Mucus+in+Histological+Sections,+Immunostaining+of+Mucins+in+Fixed+Tissue,+and+Localization+of+Bacteria+with+FISH.">Preservation of Mucus in Histological Sections, Immunostaining of Mucins in Fixed Tissue, and Localization of Bacteria with FISH.</a> Methods in Molecular Biology. 842, 229-235 (2012).</li><li>Swidsinski, A., Weber, J., Loening-Baucke, V., Hale, L. P., Lochs, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Organization+and+Composition+of+the+Mucosal+Flora+in+Patients+with+Inflammatory+Bowel+Disease.">Spatial Organization and Composition of the Mucosal Flora in Patients with Inflammatory Bowel Disease.</a> Journal of Clinical Microbiology. 43 (7), 3380-3389 (2005).</li><li>Lee, K. L., Buckley, H. R., Campbell, C. 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/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/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/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/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 ...

This protocol has yielded some of the best insights we have so far into how Candida albicans inhabits the mammalian gastrointestinal tract. For example, until recently, we didn't really know where Candida is localized within the gut or which of its many morphological forms it assumes within this niche. The main advantage of this technique is that it allows one to visualize Candida within its natural environment without disrupting the three-dimensional interactions the fungus has with host structures or with bacteria in the microbiota.Because the gut is a complex environment, it's very difficult to model in the laboratory and therefore it's been very satisfying to find a way to probe Candida biology directly in a host model. Certain components of the gut microbiota including Candida albicans are suspected to play roles in diseases such as inflammatory bowel disease. This technique can potentially be used to diagnose the presence of specific microorganisms in biopsy specimens obtained from patients.Mastery of the gavage technique and the ability to dissect gastrointestinal segments without tearing the organs is important for obtaining optimal results. To start, gavage each animal with the calculated volume of a given inoculum. Attach an autoclaved animal feeding needle to a one milliliter syringe and fill the syringe with one gavage volume making sure to remove all bubbles.Place the syringe on a sterile surface. To secure the animal, hold it at the base of the tail with the dominant hand and slide the nondominant hand up the animal's back to the loose skin just behind the ears. Gather the scruff together tightly with the thumb and forefinger.Pick up the animal by the scruff, flip it over and loop the small finger of the same hand around the tail to immobilize the animal against the palm of the hand. Gently extend the animal's head so that it is in a straight line with the body. Then pick up the syringe with the dominant hand and hold it perpendicular to the animal with the curved needle pointing down.Use the ball of the needle to gently hook an inside corner of the mouth and advance the needle along the side of the mouth to the back of the throat, then move the needle to the center. Once the ball of the needle is at the back of the animal's throat, tilt the needle up so that it is parallel with the animal's body line and allow it to slide smoothly down the throat. If the needle does not slide smoothly down the throat, the needle may not be properly positioned and should be withdrawn to reposition and try again.Smoothly advance the needle allowing the animal to swallow the needle until only a few millimeters remain visible. Test that the ball of the needle is in the stomach by gently advancing the plunger and if there is no resistance, continue delivering the inoculum. Slowly withdraw the needle once the inoculum has been administered and place the animal in its cage.Continue monitoring the animal for five to 10 minutes for signs of labored breathing or distress and verify that it resumes normal activity soon after gavage. If the same inoculum will be used for the next animal, clean the needle with an alcohol wipe. If a different inoculum will be used, replace the needle.Use blunt-ended forceps to pinch a section of the abdominal skin and incise the skin and the underlying fascia near the base of the pelvis using scissors. Extend the incision in a U-shape along each side of the peritoneal cavity and up to the ribcage, then lift the skin out of the way. Use blunt-ended forceps to gently extract the cecum from the peritoneal cavity using scissors to sever the connections between the cecum and the small and large intestines.Place the entire cecum into a histology cassette so that it is untwisted and lays flat. Place the second foam pad on top and close the cassette. Then place the cassette into a screw cap jar containing methacarn.Excise the section of the large intestines that contains one to two fecal pellets and has a length less than that of the cassette. Place the tissue into the cassette keeping it flat and untwisted. Overlay it with the second foam pad, close the cassette and place it into the methacarn.For each section of the small intestine to be sampled, excise a one to two centimeter segment that contain some digestive material. Place the tissue into the cassette, overlay with the second foam pad, close the cassette and put it into the methacarn. When excising the stomach, sever the connections to the esophagus and the small intestines.Place the tissue into the cassette and place the cassette in the methacarn. Leave the cassettes in the methacarn solution at room temperature for at least three hours but less than two weeks. After fixation, remove the foam pads and return each intact tissue section into its cassette.Then wash the tissues twice for 35 minutes in 100%methanol, twice for 25 minutes in 100%ethanol, and twice for 20 minutes in xylene. Pat the cassettes dry on a paper towel and place them in a pre-melted paraffin wax for two hours at 70 degrees Celsius in a hybridization oven. Then remove the cassettes from the wax, allow the excess wax to drain and store them at room temperature until they are embedded in wax blocks.De-wax the paraffin embedded histological sections by inserting the slides into a pre-warmed jar filled with xylene. Leave the jar at 60 degrees Celsius for 10 minutes, then discard the xylene wash. Pour fresh room temperature xylene into the jar and repeat the incubation.Then fill the jar with 100%ethanol and incubate it at room temperature for five minutes. After the incubation, remove the slides from the jar and air dry them. To stain the C.albicans with a fish probe, start by drawing a circle around the tissue section using a Pat Pen.Pipette 50 microliters of probe containing hybridization solution onto the fixed tissue and gently spread it with the pipette tip. Overlay the liquid with a hybridization coverslip making sure to prevent bubbles. Then seal the slides in a water tight hybridization chamber and incubate the sections for three hours at 50 degrees Celsius in a hybridization oven in the dark.After the incubation, add the washing solution to a Coplin jar. Carefully remove the coverslip from the slide with forceps and place the slide in the washing solution. Cover the jar with aluminum foil and incubate it at 50 degrees Celsius for 20 minutes.Next, wash the slides by pouring off the washing solution and refilling the jar with PBS. Immediately pour off and refill with fresh PBS repeating the process for a total of two washes. Wick away the PBS with a delicate task wipe and circle the intestinal tissue section with a Pat Pen.Place the slides in an opaque plastic container and add 50 microliters of mucin nuclei staining solution to the tissue section. Cover the container with aluminum foil and incubate it at four degrees Celsius for 45 minutes. After the incubation, put the slides in a Coplin jar and wash them twice with PBS.Tap away the excess PBS on a paper towel and then wick away the final drops with a tissue. Place one drop of mounting medium on each section and overlay it with a glass coverslip making sure to prevent bubbles. Let the mounting medium spread under the entire coverslip.Anchor the coverslip in place with nail polish at the corners and image the slides using a fluorescent microscope. This protocol can be used to fluorescently label C.albicans in vitro and in host tissues. In vitro propagated hyphae, round yeast cells, gut cells, and opaque cells were fixed, permeabilized, and distinguished with fluorescence and phase contrast microscopy.Round yeasts and highly elongated hyphae were imaged in the mouse large intestines. Host tissues were stained with the C.albicans specific probe or the panfungal probe. C.albicans was labeled red, host cell nuclei blue, and the mucus layer green.C.albicans was detected 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. For scientists who have not previously performed the gavage technique, it is important to obtain specialized training before attempting this method. Following fish staining, immunofluorescent techniques can be used to stain additional features of the host mucosa.This can allow for characterization of tissue damage or immune responses of the host. This technique has allowed us to characterize the relationship between fungal cell morphology of Candida albicans mutants and survival within the host furthering our understanding of Candida albicans biology that can be taken advantage of when developing therapies.

Research

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Immunology and Infection

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