in situ hybridization -s/n ratio problem
mogilvie at my-deja.com
mogilvie at my-deja.com
Mon Aug 21 11:31:08 EST 2000
Without knowing much about tissue type, etc... It sounds to me like
your problem could be in one of two places -- fixation (over which you
have no control) or endogenous biotin causing a very high background.
You can reduce endogenous background with one or two methods or you can
use a different detection system. Fluorescence is simplest if you
think your message is high enough in abundance to be seen.
Let me know if you want the protocols for reducing endogenous biotin.
Martha K. Ogilvie, Ph.D.
Scientist, Applications Laboratory
Technical Support and Customer Training
9800 Medical Center Dr.
Rockville, MD 20850
In article <04c6bcff.400e8669 at usw-ex0109-069.remarq.com>,
student <sfreckNOsfSPAM at pacbell.net.invalid> wrote:
> 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
> 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
> Link 2 (goat anti mouse IgG) RT 20min, wash (3x) PBS
> 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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