Dear Shauna
I pressure cook my sections for 1 min at full pressure followed by 45 secs
incubation (a few drops on slide) of Dako Pronase solution( follow instruction
to make up) heated for 15 minutes at 37 C. I use a 1:50 dilution of Dako T4
collagen and I get excellent results. If you over incubate in pronase your
section will get chewed up and will not stain with Haematoxylin. But 45 secs is
sufficient . I do this for other antibodies as well and get consistant staining.

David Grehan


Shauna McCabe wrote:

> Hi!
>
> I am looking for some suggestions, here is my problem:
>
> I have been doing immunostains (using ABC method) for collagen IV in mouse
> mammary tissue fixed in 10% formalin and embedded in paraffin blocks. Slides
> are cut in thin sections, are deparaffinized and rehydrated, and placed in
> 3% hydrogen peroxide to eliminate endogenous peroxidase. Following this,
> they are digested in for 1 hour  0.01% pepsin.  The pepsin digestion is
> coupled with antigen retrieval. This is where the problem lies. Through a
> test battery, it was determined that the best antigen retrieval method was
> to use a microwave pressure cooker with the retrieval solution being citrate
> buffer pH 6.0 (Pharmingen recipe). The slides are left under pressure in the
> microwave for approximately 10 minutes, and then sit in the antigen
> retrieval solution approximately 8-10 minutes longer while the pressure in
> the cooker drops.
>
> With this method, the immuno- stain is usually dark and intense and the
> counterstain (hematoxylin) either will not stain or gives uneven staining.
> The usually in last sentence is the problem. No matter how rigidly the
> protocol is followed, results are never exactly the same twice. Although the
> immuno-stain is great most of the time, it is sometimes weak or non existent
> in follow up experiments. The counterstain just doesn't want to co-operate
> at all. If the pepsin digestion step is eliminated, no collagen IV is
> detected and if it is increased in length or concentration the tissue is
> 'eaten' away.
>
> Any comments or suggestions in this matter would be greatly appreciated.
>
> Thanks,
>
>           Shauna  McCabe
>
> Co-op research Technician
> Glycodesign Inc.
> Toronto On.