[Histonet] Trypsin versus other AR-s

[Jan Shivers]

Hi Naira,

What one uses to unmask an epitope is no one answer.  When optimizing a new 
antibody, you should be trying all the various kinds of ARs available and 
comparing the results.  It's all dependent upon the antigen, where it's 
located, how difficult it is to unmask, etc.  Some methods of antigen 
retrieval actually can damage your epitope and make it unavailable for 
detection.

When I get a new Ab in, I try multiple dilutions with:
-No pretreatment
-Enzyme digestion (I use Proteinase K, but have used the other enzymes in 
the past)
-HIER (autoclave or microwave) with citrate buffer pH 6.0
-HIER (autoclave or microwave) with Target Retrieval Solution containing 
EDTA
-HIER (autoclave or microwave) with Target Retrieval Solution at pH 9.9

There are other methods which can be employed as well.  (One DOES have to 
draw the line somewhere!)

There's no set consistency between similar types of antigens either, as to 
what AR they would need.  For instance, I stain for maybe 20 different 
viruses.  Some require enzyme digestion, some require HIER (with different 
kinds of retrieval solutions).  But I only figure this out by trying all of 
the possibilities and comparing.  I would not solely depend upon the AR 
recommendations stated in Ab data sheets.  Try them all; you may have better 
results with some other type of AR than they suggest (I have).

So, don't limit your lab's AR to one type.  You could be missing much 
detection of antigen by using the wrong AR method.  However, it does require 
you to multitask, if your lab has a heavy workload and you have to perform 
AR outside of the autostainer.  Most days I'm running HIER with 3 different 
types of retrieval solutions, as well as doing enzyme digestion at the same 
time on other slides.

Jan Shivers
U of MN Vet Diag Lab

----- Original Message ----- 
From: "Margaryan, Naira" <NMargaryan <@t> childrensmemorial.org>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Friday, March 27, 2009 2:38 PM
Subject: [Histonet] Trypsin versus other AR-s


Hi Dears,



It is Friday, but not a weekend yet:-):-(



I just got a request from my PI to figure out:

1.      How often now day's people use Trypsin (EDTA, Proteinase K or E)
as an Antigen Retrieval for FFPE.

2.      Why or is the Citrate Buffer pH6 more usable???

3.      Is Trypsin very old technique?



Any educational feedback is appreciated,

Naira



_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet