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.