Hi Shauna,
    I usually avoid compound retrieval techniques. It has been my experience
that this is very harsh on the tissues, the results are usually less consistent,
and there are more opportunities for error. Collagen 4 works quite well with
proteinase K enzyme digestion. I think the heat retrieval is probably more than
the tissue needs, which would cause the excessive staining. If you don't have
proteinase K (PK) you could use another enzyme (trypsin, pepsin etc) but when we
worked this antibody up the slides stained with PK were clearly better than the
others.
good luck
Amos Brooks

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.