[Histonet] Proteolytic enzyme pretreatment before immunostaining

[William Chappell]

Enzyme pretreatment, and all steps in epitope retrieval, should follow the same quality control steps as antibody incubation and antibody concentration.

Enzyme pretreatment is not magic, however the kinetics involved are very difficult to predict.  Epitopes are typically chemical "shapes" within tertiary protein structure, however they can involve secondary structure and quartinary structure.  The purpose of enzyme pretreatment is to get the right epitope in the right configuration so it can be recognized by the antibody.  Heat retrieval is meant to break formalin cross linkages, but enzyme pretreatment actually eats away at part of the protein (depending on the protease).

Too little protease treatment and it does nothing, too much and the epitope is destroyed.

The bottom line, novel antibodies need to be validated with numerous retrieval methods.  If it is deemed that a protease is better, numerous times and numerous concentrations should be tried -- even at different temperatures.  Finally, there is no reason to believe that different novel antibodies will require the same pretreatment, however, a  common pretreatment may be sufficient (though possibly not optimal) for each antibody.

One last thing, if you know more about the nature of the antigen used to create the antibody, you may be able to predict the required pretreatment -- talk to the primary investigator.

Will Chappell, HTL(ASCP)QIHC
Anatomic Pathology Supervisor
Children's Hospital of Orange County

On Jul 31, 2012, at 8:41 PM, Young Kwun wrote:

> Dear Histonetters,
> 
> Could anyone explain about the difference between proteolytic enzymes
> for immuostaining? 
> 
> We use enzyme pretreatment rarely nowadays, and apart from some
> ready-to-use one (Dako's Proteinase K), I have used Protease (Sigma)
> previously when I did manual staining.
> 
> At the moment I am using Leica's BondMax autostainer and their enzyme
> pretreatment kit (Trypsin ??, usually one drop per 7 ml vial). I know
> the pretreatment condition would be affected by the concentration of
> enzymes, pH, temperature, incubation time etc.
> 
> 
> 
> My question is that do they have different mode of action on tissues? I
> am helping a research project and our antibody includes various clones
> of Integrin 6 subunit and uPAR.
> 
> I have tried enzyme pretreatment with autostainer and manual staining
> with Proteinase K (Dako). It seems that some antibodies work better with
> certain enzymes. 
> 
> I mean that some antibodies work well after BondMax enzyme treatment,
> but some antibody works better with proteinase K pretreatment manually.
> I am using the same polymer detection system (Leica Microsystem) for
> both methods.
> 
> I would like to find an enzyme which works for both of our antibodies at
> the same time.
> 
> Thank you.
> 
> 
> 
> 
> 
> 
> 
> Young Kwun
> 
> Senior Hospital Scientist
> 
> Immunohistochemistry
> 
> Dept. of Anatomical Pathology
> 
> Concord Repatriation General Hospital
> 
> Concord NSW 2139 Australia
> 
> 
> 
> 02-9767-6075 (Tel)
> 
> 02-9767-8427 (Fax)
> 
> Young.Kwun <@t> sswahs.nsw.gov.au
> 
> 
> 
> 
> 
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