|From:||"Tan, MinHan" |
I think that the appropriate thing to do for increased background, rather than attempt all kinds of methods, is to elicit the step at which problems are=20occurring by use of appropriate controls. This will save you both time and money ultimately.
You can run all these in a single experiment – just make sure that the slides are cut from the same specimen, which is known to stain positive.
There are quite a few causes of increased background, of course – but this will allow you to find out with the minimum of fuss, and within one day.
Slide with substrate alone
Slide with substrate + ABC
Slide with substrate + ABC + secondary antibody
Slide with substrate + ABC + primary antibody + secondary antibody
Slide with substrate + ABC + primary antibody + secondary antibody + peroxidase quenching
Slide with substrate + ABC + primary antibody + secondary antibody + blocking reageants
Slide with substrate + ABC + primary antibody + secondary antibody + peroxidase quench + blocking reageants. (dissimilar species serum)
Van Andel Research Institute
From: Scholz, Stephen J. [mailto:Stephen.J.Scholz@osfhealthcare.org]
Sent: Friday, December 05,=202003 12:32 PM
Subject: [Histonet] pretreatment for IHC
I am having a problem with background staining on IHC slides. This is only happening in cell block specimens. Is this common on cell blocks? Should I do a pretreatment on these? What pretreatments do others prefer? Should I peroxide quench? Before or after? What about blockers? Do I ramble on and ask to many questions?
Thanks for all your help,
Stephen J. Scholz HT(ASCP)
OSF St. Anthony Medical Center