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.