PMID- 22976352
OWN - NLM
STAT- MEDLINE
DCOM- 20130128
LR  - 20211021
IS  - 1750-2799 (Electronic)
IS  - 1754-2189 (Print)
IS  - 1750-2799 (Linking)
VI  - 7
IP  - 10
DP  - 2012 Oct
TI  - Dual fluorescence detection of protein and RNA in Drosophila tissues.
PG  - 1808-17
LID - 10.1038/nprot.2012.105 [doi]
AB  - Detection of RNAs by in situ hybridization (ISH) is a well-established technique 
      that permits the study of specific RNA expression patterns in tissues; however, 
      not all tissues are equally amenable to staining using the same procedure. Here 
      we describe a protocol that combines whole-mount immunofluorescence (IF) and 
      fluorescence in situ hybridization (FISH) for the simultaneous detection of 
      specific RNA transcripts and proteins, greatly enhancing the spatial resolution 
      of RNA expression in complex, intact fly tissues. To date, we have successfully 
      used this protocol in adult testis, larval male gonads, adult intestine and 
      Malpighian tubules. IF is conducted in RNase-free solutions, prior to the harsh 
      conditions of FISH, in order to preserve protein antigenicity within dissected 
      tissues. Separate protocols are described for mRNA and miRNA detection, which are 
      based on robust digoxigenin (DIG) RNA and locked nucleic acid (LNA) probes, 
      respectively. The combined IF-FISH procedure can be completed in 2 d for miRNA 
      detection and 4 d for mRNA detection. Although optimized for Drosophila, this 
      IF-FISH protocol should be adaptable to a wide variety of organisms, tissues, 
      antibodies and probes, thus providing a reliable and simple means to compare RNA 
      and protein abundance and localization.
FAU - Toledano, Hila
AU  - Toledano H
AD  - Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, 
      California, USA. hila@sci.haifa.ac.il
FAU - D'Alterio, Cecilia
AU  - D'Alterio C
FAU - Loza-Coll, Mariano
AU  - Loza-Coll M
FAU - Jones, D Leanne
AU  - Jones DL
LA  - eng
GR  - R01 AG028092/AG/NIA NIH HHS/United States
GR  - R01 AG040288/AG/NIA NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20120913
PL  - England
TA  - Nat Protoc
JT  - Nature protocols
JID - 101284307
RN  - 0 (Drosophila Proteins)
RN  - 0 (MicroRNAs)
RN  - 0 (RNA, Messenger)
SB  - IM
MH  - Animals
MH  - Drosophila/genetics/metabolism
MH  - Drosophila Proteins/*analysis/genetics
MH  - Fluorescent Antibody Technique/methods
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - MicroRNAs/*analysis/metabolism
MH  - RNA, Messenger/*analysis/metabolism
PMC - PMC4821427
MID - NIHMS713313
COIS- COMPETING FINANCIAL INTERESTS The authors declare no competing financial 
      interests.
EDAT- 2012/09/15 06:00
MHDA- 2013/01/29 06:00
PMCR- 2016/04/05
CRDT- 2012/09/15 06:00
PHST- 2012/09/15 06:00 [entrez]
PHST- 2012/09/15 06:00 [pubmed]
PHST- 2013/01/29 06:00 [medline]
PHST- 2016/04/05 00:00 [pmc-release]
AID - nprot.2012.105 [pii]
AID - 10.1038/nprot.2012.105 [doi]
PST - ppublish
SO  - Nat Protoc. 2012 Oct;7(10):1808-17. doi: 10.1038/nprot.2012.105. Epub 2012 Sep 
      13.