Re: Bobrowitz-InSitu
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From: | "Tony Henwood" <henwood@mail.one.net.au> |
To: | Dana Settembre <settembr@UMDNJ.EDU>, kkdulany@unmc.edu |
Reply-To: | |
Date: | Wed, 6 Oct 1999 21:42:44 +0000 |
Content-Type: | text/plain; charset=US-ASCII |
Dear Karen,
>
> I see the in situ question has arisen again. We have still not gotten
> anywhere with this and our pathologist is trying to be patient. He wants
> us to do in situ on pancreas slides, paraffin and formalin fixed. We are
> using an oligo probe to insulin and tail it with DIG.
>
> Does anyone have a tried and true method for doing this type of in situ??
> Marianne and I don't feel qualified to do this without molecular
> experience but seems like we have to try to learn it.
I have used the following technique successfully for mRNA
demonstration. It is simple and quite robust. Pancreas may be a
problem since post mortem autolysis sets in quite quickly but lots of
luck.
Regards, Tony
IN-SITU HYBRIDISATION - mRNA TARGET
REFERENCE:
SOLUTIONS:
l. 20 x SSC l75g Sodium Chloride
88g Sodium Citrate dihydrate
Make up to 1L with deionised water
2. 2 x SSC lml 20 x SSC
dilute to l00 ml with deionised water
3. Endogenous Peroxidase block
Methanol 45ml
30% Hydrogen Peroxide 5ml
4. 0.2N Hydrochloride Acid
Concent HCl (10N) 1ml.
Distilled water 49ml.
5. Enzyme pretreatment
Dulbecco A PBS tablets 1 tablet
Pronase (Bacterial protease XXIV Sigma P8038) 50mg
Sodium Azide 100mg
Distilled water 100ml.
6. Probe working solution
I use Sigma's 2x Hybridisation solution and donot add formamide.
PROCEDURE:
1. Dewax sections in xylene, rinse in alcohol and air dry.
2. Circle sections with PAP pen or equivalent.
3. Block endogenous peroxidase 5 min rt.
4. Wash slides in water.
5. Place in 0.2N HCl at room temperature 10 min.
6. Rinse slides in water.
7. Treat sections with pronase 7 min at 42oC.
8. Rinse slides in water.
9. Dehydrate slides in alcohol and air dry.
10. Add 1-2 drops of working probe to section and gently cover section with coverslip.
11. Allow hybridisation in a humid atmosphere at room temperature over night.
12. Wash off coverslips in 2 X SCC.
13. Place slides in buffer in preparation for label demonstration.
Tony Henwood
Senior Scientist
Anatomical Pathology
Royal Prince Alfred Hospital
Sydney, AUSTRALIA
http://www2.one.net.au/~henwood
http://www.pathsearch.com/homepages/TonyHenwood/default.html
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