Hi Karen, if the antibody is clone DC-10, then antigen retrieval might work
better than protease digestion. A buffer of pH 4 gave us the best
signal-to-noise.
Abizar
www.innogenex.com

Karen Pawlowski wrote:

> Help,
>
> I am having trouble getting any staining (background or otherwise) with
> CK18 labeling. The tissue is formalin fixed, paraffin embedded rat sub-
> mandibular gland, which according to the data sheet should stain.
> I am using a Sigma antibody at the recommended dilution and twice that
> strength, for the recommended time (1 hr) and at 4C overnight. I have
> tried protease E digestion and micro waving with citrate phosphate
> buffer to no avail. I have tried the MOM kit from Vector (I wanted to
> eventually do mice) and a direct labeled secondary with no blocking.
>
> All I've seen so far is RBC staining (endogenous) on the tissue that
> I didn't first expose to H2O2.  I figure I should at least see some
> nonspecific staining of the secondary when I skip all blocking steps,
> but that doesn't happen. Any ideas? [I checked the DAB and secondary
> HRP and they seem to react with one another.]
>
> Also, we used a xylene substitute to deparaffinize (histoclear).
> Anyone had problems with using xylene substitutes with
> IHC? Am I missing some-thing obvious?
>
> I've contacted both Sigma and Vector, but so far the only thing they
> suggest that I haven't already tried was xylene deparaffinization.
>
> Karen Pawlowski, Ph.D.