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)

 

Min-Han Tan

Van Andel Research Institute

 

 

-----Original Message-----
From: Scholz, Stephen J. [mailto:Stephen.J.Scholz@osfhealthcare.org]
Sent: Friday, December 05,=202003 12:32 PM
To: Histonet@Pathology.swmed.edu
Subject: [Histonet] pretreatment for IHC

 

Hello Histonet,

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)
Histology Coordinator
OSF St. Anthony Medical Center
Rockford IL

Phone: 815-395-5410
Fax: 815-395-5364
e-mail: sjscholz@osfhealthcare.org