in situ hybridization -s/n ratio problem

student sfreckNOsfSPAM at pacbell.net.invalid
Sat Aug 19 20:02:11 EST 2000


i have been trying to perform in situ hybridization on
tissue sections using probe labelled with vector's long-arm
biotin - the problem i keep encountering is very high
background and virtually no signal - sense, antisense, and
negative controls were run on two tissue sections and
visualized by DAB and BCIP both had s/n problems - sense,
antisense and negative looked the same due to extremely
high background and lack of distinguishable signal;
biotinylated DNA was run as a control with the same tissues
and both stains and still showed the same s/n problems

has anyone else encountered this problem? suggestions on
protocol changes? (the lab i am using is not set up for
radiolabelling, but FISH is possible - non-flourescent
labelling was being used due to background problems with
FISH on paraffin embedded tissue)

any help or suggestions would be greatly appreciated.


i am using formalin fixed paraffin embedded tissue sections
cut to 6nm and floated onto suprafrost slides (tissue
sections are up to 8 yrs old - i have no control over
fixation of tissue)

biogenex in situ hybridization kit is being used

protocol as follows:
 deparaffinize (3x) xylene 3min/ea, (3x) 100% EtOH 1min/ea,
80 % EtOH 1 min, 50% EtOH with RNase Block 1min, DI H2O w/
RNase Block 1 min
 Proteinase K digestion RT 15min; wash PBS w/ RNase Block 5
min
 dehydrate DI H2O, 50% EtOH, 70% EtOH, 100% EtOH 10s/ea; dry
RT 15 min
 prehybridization solution incubated 37C 60 min; wash (3x)
100% EtOH 3min/ea, dry RT 15 min
 hybridization w/ biotinylated oligo probe (100ng/ml) heat
95C 10min, incubate 37C overnight
 wash (3x) 2XSSC 5min/ea, 1XSSC 5min
 Protein Block incubated RT 20min, wash (3x) PBS 5min/ea
 Link1 (mouse anti-biotin IgG) RT 20min, wash (3x) PBS
5min/ea
 Link 2 (goat anti mouse IgG) RT 20min, wash (3x) PBS
5min/ea
 Label incubated RT 20min, wash (3x) PBS 5min/ea, followed
by DAB 3min, and hematoxylin counterstain
(alternatively alk phos label incubated RT 30 min rinsed w/
PBS 5min, DI H2O (3x) 1min/ea, .1M Tris 5min, followed by
BCIP/NBT 30 min, and nuclear fast red counterstain



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