[Histonet] RE: Proteolytic enzyme pretreatment before immunostaining

[Goins, Tresa]

When re-reading my e-mail, it does sound like we use heat and enzymatic retrieval together - we do not - we assess the effective of all retrieval methods used singly.

Tresa

-----Original Message-----
From: Gudrun Lang [mailto:gu.lang <@t> gmx.at] 
Sent: Wednesday, August 01, 2012 10:28 AM
To: Goins, Tresa
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: AW: [Histonet] RE: Proteolytic enzyme pretreatment before immunostaining

I think formalin fixation does something similar to enzyme-"binding" as to antibody-binding, if the enzyme has a specific binding site. The aminoacids are masked by methylol-adducts and therefore the protein is protected against eg pepsin.
So using combinated HIER and PIER is not only summarizing the effects. HIER renders PIER more effective and therefore more aggressive.

Gudrun Lang

-----Ursprüngliche Nachricht-----
Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Goins, Tresa
Gesendet: Mittwoch, 01. August 2012 16:00
An: Young Kwun; histonet <@t> lists.utsouthwestern.edu
Betreff: [Histonet] RE: Proteolytic enzyme pretreatment before immunostaining

The proteolytic activity of the enzymes can be non-specific (Pronase) or specific (peptide bonds at lysine and arginine residues, Trypsin).  The effect of formalin fixation is not identified, so we always assess Pepsin, Trypsin and Pronase along with heat retrieval methods regardless of what the
supplier data sheet recommends.    

Tresa  



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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