RE: [Histonet] rat gi

From:"Elizabeth Chlipala"


Try Dako's LSAB2 kit for rat specimens.  I use it all the time for mouse
monoclonal antibodies on rat specimens.  With this kit it there is no
background staining.  I also use Dako's Serum free protein block.  Off
the histonet I'll send you some images of the same PCNA antibody on rat
gut.  I find with secondary reagents you need to check the specification
sheet very carefully before purchasing, especially if you are dealing
with animal tissue.  On the spec sheet it will tell you the species
crossreactivity.  You need to check if your secondary will cross react
with the same species of your tissue.  I have bought secondary
antibodies that I have in the fridge that I can't use (they were
recommended by the vendor of the primary antibody) but they cross react
with the tissue that I'm testing.  I learned that little tid bit the
hard way.


Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
P.O. Box 18592
Boulder, Colorado 80308
Office: (303) 735-5001
Fax: (303) 735-3540
Ship to Address:
Premier Laboratory
University of Colorado
MCDB, Room A3B40
Boulder, Colorado 80309

-----Original Message-----
[] On Behalf Of Till,
Sent: Friday, March 04, 2005 8:35 AM
Subject: [Histonet] rat gi

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).




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