PMID- 7584553
OWN - NLM
STAT- MEDLINE
DCOM- 19951214
LR  - 20191031
IS  - 0948-6143 (Print)
IS  - 0948-6143 (Linking)
VI  - 103
IP  - 6
DP  - 1995 Jun
TI  - Qualitative and quantitative detection of alkaline phosphatase coupled to an 
      oligonucleotide probe for somatostatin mRNA after in situ hybridization using 
      unfixed rat brain tissue.
PG  - 463-71
AB  - In situ hybridization (ISH) of somatostatin (SOM) mRNA was carried out on 
      sections of rat brain using an alkaline phosphatase (AP) coupled oligonucleotide 
      probe. Different hybridization and AP development conditions were tested for 
      qualitative and quantitative detection of target mRNA on sections of unfixed 
      tissue. Hybridization signal intensities after 24 h of hybridization were high. 
      Comparison with adjacent formaldehyde-fixed tissue sections and hybridization for 
      various lengths of time (2-42 h) indicated that in unfixed tissue retention of 
      SOM mRNA was at least as high as after fixation, and that the mRNA was not 
      degraded during hybridization. The use of tetranitroblue instead of nitroblue 
      tetrazolium chloride in the AP detection medium provided a superior 
      signal-to-noise ratio, and medium stability was improved for quantitative studies 
      on unfixed sections by adding 10% polyvinyl alcohol at pH 8.5. Microphotometric 
      measurements of mean optical densities (MOD) of the formazan reaction product in 
      a defined area within individual neurons of the lateral central amygdaloid 
      nucleus showed a linear increase over the first 23 h of AP reaction time. The 
      mean MOD values per neuron were comparably high in various equally thick sections 
      of the nucleus and increased with section thickness in a linear manner. The 
      findings indicate that the ISH and detection reagents penetrate the entire 
      section and that there is a linear relationship between the amount of AP reaction 
      product measured and the amount of mRNA present in the measured area. Thus, ISH 
      using an AP-coupled oligonucleotide on sections of unfixed tissue appears 
      suitable for quantitative mRNA detection.
FAU - Asan, E
AU  - Asan E
AD  - Institute of Anatomy, University of Wurzburg, Germany.
FAU - Kugler, P
AU  - Kugler P
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Germany
TA  - Histochem Cell Biol
JT  - Histochemistry and cell biology
JID - 9506663
RN  - 0 (Oligonucleotide Probes)
RN  - 0 (RNA, Messenger)
RN  - 51110-01-1 (Somatostatin)
RN  - EC 3.1.3.1 (Alkaline Phosphatase)
SB  - IM
MH  - Alkaline Phosphatase/*analysis
MH  - Animals
MH  - Brain/*enzymology
MH  - Brain Chemistry
MH  - Immunohistochemistry
MH  - In Situ Hybridization/*methods
MH  - Male
MH  - Oligonucleotide Probes
MH  - RNA, Messenger/*analysis
MH  - Rats
MH  - Rats, Wistar
MH  - Somatostatin/*genetics
MH  - Tissue Fixation
EDAT- 1995/06/01 00:00
MHDA- 1995/06/01 00:01
CRDT- 1995/06/01 00:00
PHST- 1995/06/01 00:00 [pubmed]
PHST- 1995/06/01 00:01 [medline]
PHST- 1995/06/01 00:00 [entrez]
AID - 10.1007/BF01457546 [doi]
PST - ppublish
SO  - Histochem Cell Biol. 1995 Jun;103(6):463-71. doi: 10.1007/BF01457546.