Re: [Histonet] IHC Negative reagent controls

[Tony Henwood (SCHN) via Histonet]

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

Regards 

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@lists.utsouthwestern.edu] 
Sent: Thursday, 3 January 2019 1:48 PM
To: histonet@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?

Frank

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[Histonet] IHC Negative reagent controls

[Cayman Fleck via Histonet]

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?

Frank

Sent from Outlook
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