Re: how to dehydrate slides stained with AEC
Dear Baowei,
Based on our experience, not only you should use anti-rat secondary
antibody, it also has to be absorbed with mouse serum protein to minimize
cross reaction to mouse antibodies and mouse tissue proteins. Also,
reducing secondary and enzyme incubation time to 5 minutes each should be
sufficient for cryostat sections.
AEC stains can be stored for many years in dry condition. We do the
following:
Counterstain with hematoxylin and wash with PBS or TBS
Rinse with dd water (or soak with dd water for a feww dips)
Mount with water based mounting media (available from many sources)
Incubate at 65 oC for 30 minute to dry
Store in dark
Good luck
James Guo
ImmunoVision Technologies, Co.
www.immunovisiontech.com
At 10:09 PM 7/27/2003 +0800, you wrote:
Dear Kiernan,
Thank you for your advise.
I had modified my experimental strategy. I used mouse spleen to test the
system since I was intended to indentify CD4 cell. I did it as following
on acetone fixed frozen section:
(1) negative control was done with primary antibody being omitted
(2) positive control was included too.
(3) primary antibody, 30 min
(4) 2nd antibody, 30 min
(5) Enzyme, 30 min
(6) AEC substrate, 6 min
(7) hematoxylin conterstain, 5 min
There was red stain on negative control slide but not all the silde while
the positive was all stained red very where. I noticed the 2nd antibody
was antimouse. But my primary antibody was take from rat. Must I use 2nd
anti-rat antiboy?
Baowei Peng
Shanghai JiaoTong University,
Shanghai,200030
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