[Histonet] IHC Negative reagent controls

Tony Henwood (SCHN) tony.henwood at health.nsw.gov.au
Wed Jan 2 22:06:57 CST 2019

Hi Cayman,

Unfortunately, applying HIER to a negative control for an antibody that requires enzyme retrieval (or no retrieval at all) is not appropriate.
The pre-treatment processes are different and could unmask different epitopes.
If you are using a negative control then the whole procedure needs to be same with the exception or replacing the localisation antibody with an Isotypic antibody solution. (Isotype controls are primary antibodies that lack specificity to the target, but match the class and type of the primary antibody used in the application.)

For example, applying citrate or EDTA HIER to sections prior to using the CD99 antibody (clone 12E7) can reveal perinuclear (golgi-like) staining of some tumours (eg some colonic carcinomas) but this is not seen if enzyme retrieval is used.

Hope this is useful


Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: Cayman Fleck via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Thursday, 3 January 2019 1:48 PM
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] IHC Negative reagent controls

A question that came up regarding negative reagent controls for IHC...currently using Ventana i-View.  Our regular negative control goes through the standard antigen retrieval steps, like 99% of our antibodies.  However there are a small number of antibodies that require enzyme as well (Protease 1).  I've seen a number of suggestions regarding this for the negative reagent control...some say use an additional negative control protocol that includes the protease, some say to use a single negative control protocol and just include the harshest cell conditioning that any of your protocols use (so basically use the cell conditioning + protease negative control for all antibodies)...i-View is not polymer-based so we need to continue using negative controls.  Any thoughts or advice?


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