PMID- 11101627
OWN - NLM
STAT- MEDLINE
DCOM- 20010118
LR  - 20170214
IS  - 0022-1554 (Print)
IS  - 0022-1554 (Linking)
VI  - 48
IP  - 12
DP  - 2000 Dec
TI  - Fluorescence in situ hybridization of scarce leptin receptor mRNA using the 
      enzyme-labeled fluorescent substrate method and tyramide signal amplification.
PG  - 1593-99
AB  - To increase the sensitivity of fluorescence in situ hybridization (FISH) for 
      detection of low-abundance mRNAs, we performed FISH on cryostat sections of rat 
      hypothalamus with biotin-labeled riboprobes to leptin receptor (ObRb) and 
      amplified the signal by combining tyramide signal amplification (TSA) and 
      Enzyme-Labeled Fluorescent alkaline phosphatase substrate (ELF) methods. First, 
      TSA amplification was done with biotinylated tyramide. Second, 
      streptavidin-alkaline phosphatase was followed by the ELF substrate, producing a 
      bright green fluorescent reaction product. FISH signal for ObRb was undetectable 
      when TSA or ELF methods were used alone, but intense ELF FISH signal was visible 
      in hypothalamic neurons when the ELF protocol was preceded by TSA. The TSA-ELF 
      was combined with FISH for pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) 
      mRNAs by hybridizing brain sections in a cocktail containing digoxigenin-labeled 
      riboprobes to NPY or POMC mRNA and biotin-labeled riboprobes to ObRb mRNA. 
      Dioxigenin-labeled NPY or POMC mRNA hybrids were subsequently detected first with 
      IgG-Cy3. Then biotin-labeled leptin receptor hybrids were detected with the 
      TSA-ELF method. Combining the ELF and TSA amplification techniques enabled FISH 
      detection of scarce leptin receptor mRNAs and permitted the identification of 
      leptin receptor mRNA in cells that also express NPY and POMC gene products.
FAU - Breininger, J F
AU  - Breininger JF
AD  - Division of Endocrinology/Metabolism, Medical Research Service, VA Puget Sound 
      Health Care System, and Departments of Medicine and Biological Structure, 
      University of Washington School of Medicine, Seattle, Washington 98108, USA.
FAU - Baskin, D G
AU  - Baskin DG
LA  - eng
GR  - DK17047/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Histochem Cytochem
JT  - The journal of histochemistry and cytochemistry : official journal of the 
      Histochemistry Society
JID - 9815334
RN  - 0 (Carrier Proteins)
RN  - 0 (Leptin)
RN  - 0 (RNA, Messenger)
RN  - 0 (Receptors, Cell Surface)
RN  - 0 (Receptors, Leptin)
RN  - 0 (biotinyltyramide)
RN  - 6SO6U10H04 (Biotin)
RN  - 9013-20-1 (Streptavidin)
RN  - EC 3.1.3.1 (Alkaline Phosphatase)
RN  - X8ZC7V0OX3 (Tyramine)
SB  - IM
MH  - Alkaline Phosphatase
MH  - Animals
MH  - Biotin/*analogs & derivatives
MH  - Carrier Proteins/*analysis/metabolism
MH  - Hypothalamus/metabolism
MH  - In Situ Hybridization, Fluorescence/methods
MH  - Leptin/*metabolism
MH  - Male
MH  - Microscopy, Fluorescence
MH  - RNA, Messenger/*analysis
MH  - Rats
MH  - Rats, Wistar
MH  - *Receptors, Cell Surface
MH  - Receptors, Leptin
MH  - Streptavidin
MH  - Tyramine/*analogs & derivatives
EDAT- 2000/12/02 11:00
MHDA- 2001/02/28 10:01
CRDT- 2000/12/02 11:00
PHST- 2000/12/02 11:00 [pubmed]
PHST- 2001/02/28 10:01 [medline]
PHST- 2000/12/02 11:00 [entrez]
AID - 10.1177/002215540004801202 [doi]
PST - ppublish
SO  - J Histochem Cytochem. 2000 Dec;48(12):1593-99. doi: 10.1177/002215540004801202.