[Histonet] rat gi

From:"Till, Renee"

Does anyone have any suggestions for reducing background with rat gi? I
have been trying to do a PCNA stain on distal and proximal colon with
varying results. We use M0879 PC10 PCNA from Dako, which is a monoclonal
mouse antibody, along with Biotin Conjugated Affinity Purified Goat
anti-Mouse IgG from Rockland, and Peroxidase Conjugated Streptavidin
from Jackson. I block with CAS block from Zymed and antigen retrieve
with CitraPlus from Biogenex. I have also just tried using the
Vectastain ABC Mouse kit, but that seems to turn out even worse than
when I make my link and label. Could it be something as simple as longer
washes or different antigen retrieval? Is it that important that you let
the slides cool for the 20-30 mintues after retrieval as opposed to
continuing right away or cooling them yourself? Or is gi just a tissue
that is always going to give me background problems. Or could it be the
antibody itself? I will admit I don't know enough about the specifics of
how immunos work to figure out exactly what it is that could be causing
the background staining.  So far I have just tried to get them stained
as best as possible without the background staining interfering with
with my counting ( I have to count crypts cells by the intensity of
staining, which is supposed to relate to the cell phase).

 

Thanks,

Renee' 


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