PMID- 35969095
OWN - NLM
STAT- MEDLINE
DCOM- 20220817
LR  - 20230301
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 185
DP  - 2022 Jul 27
TI  - RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization 
      and Infection in Caenorhabditis elegans Intestines.
LID - 10.3791/63980 [doi]
AB  - The intestines of wild Caenorhabditis nematodes are inhabited by a variety of 
      microorganisms, including gut microbiome bacteria and pathogens, such as 
      microsporidia and viruses. Because of the similarities between Caenorhabditis 
      elegans and mammalian intestinal cells, as well as the power of the C. elegans 
      system, this host has emerged as a model system to study host intestine-microbe 
      interactions in vivo. While it is possible to observe some aspects of these 
      interactions with bright-field microscopy, it is difficult to accurately classify 
      microbes and characterize the extent of colonization or infection without more 
      precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a 
      tool to identify and visualize microbes in nematodes from the wild or to 
      experimentally characterize and quantify infection in nematodes infected with 
      microbes in the lab. FISH probes, labeling the highly abundant small subunit 
      ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. 
      Probes designed to target conserved regions of ribosomal RNA common to many 
      species can detect a broad range of microbes, whereas targeting divergent regions 
      of the ribosomal RNA is useful for narrower detection. Similarly, probes can be 
      designed to label viral RNA. A protocol for RNA FISH staining with either 
      paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal 
      for nematodes associated with bacteria, microsporidia, and viruses, whereas 
      acetone fixation is necessary for the visualization of microsporida spores. 
      Animals were first washed and fixed in paraformaldehyde or acetone. After 
      fixation, FISH probes were incubated with samples to allow for the hybridization 
      of probes to the desired target. The animals were again washed and then examined 
      on microscope slides or using automated approaches. Overall, this FISH protocol 
      enables detection, identification, and quantification of the microbes that 
      inhabit the C. elegans intestine, including microbes for which there are no 
      genetic tools available.
FAU - Rivera, Dalaena E
AU  - Rivera DE
AD  - Department of Biology, San Diego State University.
FAU - Lazetic, Vladimir
AU  - Lazetic V
AD  - School of Biological Sciences, University of California, San Diego.
FAU - Troemel, Emily R
AU  - Troemel ER
AD  - School of Biological Sciences, University of California, San Diego.
FAU - Luallen, Robert J
AU  - Luallen RJ
AD  - Department of Biology, San Diego State University; rluallen@sdsu.edu.
LA  - eng
GR  - R01 AG052622/AG/NIA NIH HHS/United States
GR  - R01 GM114139/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PT  - Video-Audio Media
DEP - 20220727
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 0 (RNA, Ribosomal)
RN  - 1364PS73AF (Acetone)
RN  - 63231-63-0 (RNA)
SB  - IM
MH  - Acetone
MH  - Animals
MH  - Bacteria/genetics
MH  - *Caenorhabditis
MH  - Caenorhabditis elegans/genetics/microbiology
MH  - In Situ Hybridization, Fluorescence
MH  - Intestines/microbiology
MH  - Mammals/genetics
MH  - *Microsporidia/genetics
MH  - RNA
MH  - RNA, Ribosomal
MH  - *Viruses/genetics
PMC - PMC9969837
MID - NIHMS1873725
COIS- Disclosures The authors have no conflicts of interest.
EDAT- 2022/08/16 06:00
MHDA- 2022/08/18 06:00
PMCR- 2023/02/27
CRDT- 2022/08/15 09:05
PHST- 2022/08/15 09:05 [entrez]
PHST- 2022/08/16 06:00 [pubmed]
PHST- 2022/08/18 06:00 [medline]
PHST- 2023/02/27 00:00 [pmc-release]
AID - 10.3791/63980 [doi]
PST - epublish
SO  - J Vis Exp. 2022 Jul 27;(185):10.3791/63980. doi: 10.3791/63980.