[Histonet] Re: troubles with CDK4
I can't find a Cdk4 (D-22) antibody in their catalog. I see a C-22 and
an H-22. If you are using the C-22, which they indicate can be used in
IHC-paraffin embedded material, call them and ask what their staining
conditions are (including fixative used).
The detection reagents you are using might be ultra sensitive (too
sensitive) for this antibody. Either extend the dilution of the primary
antibody, or try using an HRP conjugated anti-rabbit secondary antibody
at the concentration you are currently using to see if that lowers the
background staining. The staining intensity in the picture in the Santa
Cruz catalog of the C-22 on human skin is very weak. Perhaps that's the
best signal they can get with this antibody while keeping the background
noise in check.
You might also try pre-blocking the primary antibody by adding 2-5%
serum concentration of the species you are staining to the antibody
diluent you are using. Make your antibody dilution in this and let it
incubate in the tube prior to placing it on the slide.
There are a variety of things you can try, but be aware that you may
never get it working suitably despite the company's claims. You might
need to look elsewhere for a different source. Gayle Callis turned me on
to this site: http://www.exactantigen.com/ and another good one is
Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64133
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