in situ hybridization -s/n ratio problem

Robert Hartley rh at mblab.gla.ac.uk
Mon Aug 21 06:08:55 EST 2000


In article <04c6bcff.400e8669 at usw-ex0109-069.remarq.com>, student
<sfreckNOsfSPAM at pacbell.net.invalid> wrote:


There are lots of modifications to various protocols.It really depends
what the target is. This might not be very helpfull.

BUT

I would recomend getting scouring the library for this book or contacting
Boehringer Mannheim to see if they still supply it. IMHO no lab should be
without it.

" Nonradioactive in-situ hybridisation. Aplication manual.secpond ed"

No ISBN so it is probably a freebie.

Cheers
Bob

> 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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