Re: Bobrowitz-InSitu

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From:"Tony Henwood" <>
To:Dana Settembre <settembr@UMDNJ.EDU>,
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 
Regards, Tony




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.

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

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