PMID- 29176788
OWN - NLM
STAT- MEDLINE
DCOM- 20171211
LR  - 20201214
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 12
IP  - 11
DP  - 2017
TI  - Preservation of three-dimensional spatial structure in the gut microbiome.
PG  - e0188257
LID - 10.1371/journal.pone.0188257 [doi]
LID - e0188257
AB  - Preservation of three-dimensional structure in the gut is necessary in order to 
      analyze the spatial organization of the gut microbiota and gut luminal contents. 
      In this study, we evaluated preparation methods for mouse gut with the goal of 
      preserving micron-scale spatial structure while performing fluorescence imaging 
      assays. Our evaluation of embedding methods showed that commonly used media such 
      as Tissue-Tek Optimal Cutting Temperature (OCT) compound, paraffin, and polyester 
      waxes resulted in redistribution of luminal contents. By contrast, a hydrophilic 
      methacrylate resin, Technovit H8100, preserved three-dimensional organization. 
      Our mouse intestinal preparation protocol optimized using the Technovit H8100 
      embedding method was compatible with microbial fluorescence in situ hybridization 
      (FISH) and other labeling techniques, including immunostaining and staining with 
      both wheat germ agglutinin (WGA) and 4', 6-diamidino-2-phenylindole (DAPI). Mucus 
      could be visualized whether the sample was fixed with paraformaldehyde (PFA) or 
      with Carnoy's fixative. The protocol optimized in this study enabled simultaneous 
      visualization of micron-scale spatial patterns formed by microbial cells in the 
      mouse intestines along with biogeographical landmarks such as host-derived mucus 
      and food particles.
FAU - Hasegawa, Yuko
AU  - Hasegawa Y
AD  - Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine 
      Biological Laboratory, Woods Hole, Massachusetts, United States of America.
FAU - Mark Welch, Jessica L
AU  - Mark Welch JL
AUID- ORCID: 0000-0003-0696-2334
AD  - Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine 
      Biological Laboratory, Woods Hole, Massachusetts, United States of America.
FAU - Rossetti, Blair J
AU  - Rossetti BJ
AD  - Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine 
      Biological Laboratory, Woods Hole, Massachusetts, United States of America.
AD  - Department of Microbiology, The Forsyth Institute, Cambridge, Massachusetts, 
      United States of America.
FAU - Borisy, Gary G
AU  - Borisy GG
AD  - Department of Microbiology, The Forsyth Institute, Cambridge, Massachusetts, 
      United States of America.
LA  - eng
GR  - R01 DE022586/DE/NIDCR NIH HHS/United States
PT  - Journal Article
DEP - 20171127
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (Methacrylates)
RN  - 0 (Polymers)
RN  - 1HG84L3525 (Formaldehyde)
RN  - 9012-36-6 (Sepharose)
RN  - Y19UC83H8E (paraform)
SB  - IM
MH  - Animals
MH  - Cryoultramicrotomy
MH  - Formaldehyde/chemistry
MH  - *Gastrointestinal Microbiome
MH  - Germ-Free Life
MH  - In Situ Hybridization, Fluorescence
MH  - Methacrylates
MH  - Mice, Inbred C57BL
MH  - Mucus/metabolism
MH  - Paraffin Embedding
MH  - Polymers/chemistry
MH  - *Preservation, Biological
MH  - Sepharose
MH  - Tissue Fixation
PMC - PMC5703541
COIS- Competing Interests: The authors have declared that no competing interests exist.
EDAT- 2017/11/28 06:00
MHDA- 2017/12/12 06:00
PMCR- 2017/11/27
CRDT- 2017/11/28 06:00
PHST- 2017/08/22 00:00 [received]
PHST- 2017/11/05 00:00 [accepted]
PHST- 2017/11/28 06:00 [entrez]
PHST- 2017/11/28 06:00 [pubmed]
PHST- 2017/12/12 06:00 [medline]
PHST- 2017/11/27 00:00 [pmc-release]
AID - PONE-D-17-30893 [pii]
AID - 10.1371/journal.pone.0188257 [doi]
PST - epublish
SO  - PLoS One. 2017 Nov 27;12(11):e0188257. doi: 10.1371/journal.pone.0188257. 
      eCollection 2017.