PMID- 32908315
OWN - NLM
STAT- MEDLINE
DCOM- 20201103
LR  - 20210810
IS  - 1750-2799 (Electronic)
IS  - 1750-2799 (Linking)
VI  - 15
IP  - 10
DP  - 2020 Oct
TI  - Combined whole-mount fluorescence in situ hybridization and antibody staining in 
      zebrafish embryos and larvae.
PG  - 3361-3379
LID - 10.1038/s41596-020-0376-7 [doi]
AB  - RNA fluorescence in situ hybridization (FISH) and antibody 
      staining/immunofluorescence (IF) are widely used to detect distributions of mRNAs 
      and proteins. Here we describe a combined FISH and IF protocol to simultaneously 
      detect multiple mRNAs and proteins in whole-mount zebrafish embryos and larvae. 
      In our approach, FISH is performed before IF to prevent mRNA degradation during 
      the IF procedure. Instead of proteinase K digestion, Triton X-100 treatment and 
      skin removal are used to permeate tissues and preserve antigen epitopes, making 
      this protocol applicable to both whole-mount embryos and larvae. Off-target 
      hybridization and FISH background are reduced by using PCR-amplified DNA 
      templates and stringent buffers. This protocol simultaneously detects multiple 
      mRNAs and proteins with high sensitivity, and enables detection at single-cell 
      resolution. The protocol can be completed within 6 days, overcoming the shortage 
      of reliable antibodies available for zebrafish and exploiting the advantages of 
      zebrafish for studying organ development and regeneration.
FAU - He, Jianbo
AU  - He J
AD  - Institute of Developmental Biology and Regenerative Medicine, Southwest 
      University, Beibei, Chongqing, China.
FAU - Mo, Dashuang
AU  - Mo D
AD  - Institute of Developmental Biology and Regenerative Medicine, Southwest 
      University, Beibei, Chongqing, China.
FAU - Chen, Jingying
AU  - Chen J
AD  - Institute of Developmental Biology and Regenerative Medicine, Southwest 
      University, Beibei, Chongqing, China.
FAU - Luo, Lingfei
AU  - Luo L
AUID- ORCID: 0000-0002-5318-8137
AD  - Institute of Developmental Biology and Regenerative Medicine, Southwest 
      University, Beibei, Chongqing, China. lluo@swu.edu.cn.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20200909
PL  - England
TA  - Nat Protoc
JT  - Nature protocols
JID - 101284307
RN  - 0 (Antibodies)
RN  - 0 (RNA, Messenger)
RN  - 63231-63-0 (RNA)
SB  - IM
MH  - Animals
MH  - Antibodies/metabolism
MH  - Embryo, Nonmammalian/*diagnostic imaging
MH  - Fluorescent Antibody Technique/*methods
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Larva/metabolism
MH  - RNA/metabolism
MH  - RNA, Messenger/genetics
MH  - Zebrafish/genetics
EDAT- 2020/09/11 06:00
MHDA- 2020/11/04 06:00
CRDT- 2020/09/10 05:34
PHST- 2019/04/25 00:00 [received]
PHST- 2020/06/19 00:00 [accepted]
PHST- 2020/09/11 06:00 [pubmed]
PHST- 2020/11/04 06:00 [medline]
PHST- 2020/09/10 05:34 [entrez]
AID - 10.1038/s41596-020-0376-7 [pii]
AID - 10.1038/s41596-020-0376-7 [doi]
PST - ppublish
SO  - Nat Protoc. 2020 Oct;15(10):3361-3379. doi: 10.1038/s41596-020-0376-7. Epub 2020 
      Sep 9.