[Histonet] Proteolytic enzyme pretreatment before immunostaining

[Young Kwun]

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