Re: Bobrowitz-InSitu

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From:"Tony Henwood" <henwood@mail.one.net.au>
To:Dana Settembre <settembr@UMDNJ.EDU>, kkdulany@unmc.edu
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Date:Wed, 6 Oct 1999 21:42:44 +0000
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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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