[Histonet] RE: IHC Doublestain

[C.M. van der Loos]

Kim,This is a difficult one! First HIER and then prot K may destroy/remove antigens, not to speak about the integrity of the tissue morphology! The best you can do is first performing HIER and then prot K only half of the time. Again, it fully depends on the antigens of interest if both will be still there after this harsh treatment. If you are using a DAKO sequential double staining method you may start with HIER followed by the 1st primary antibody (the one that needs HIER), detection system, finish with DAB in brown, then prot K (half of the time) followed by the 2nd primary, detection sytem, AP in red. Alternatively, you can follow the same sequential procedure with two AP methods as we described in JOH of last September. PDF is send to your work address.  The second best I can offer you is to find a HIER method that works for both antigens. HIER with Tris/EDTA pH9.0 is effective for many antigens that are usually enzymatically pretreated. Good luck!ChrisChris van der Lo!
 os, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands Date: Wed, 07 Jan 2009 11:21:13 -0500
From: "Kimberly Tuttle" <ktuttle <@t> umm.edu>
Subject: [Histonet] IHC Doublestain
To: histonet <@t> lists.utsouthwestern.edu

I am supposed to try a  doublestain using the DAKO autostainer where one of the antibodies requires a Proteinase K pretreatment and the other antibody requires antigen retrieval with heat.  Should I do both, or will this not work at all? Thank you

Kimberly C. Tuttle  HT (ASCP)
Pathology Biorepository and Research Core
University of Maryland 
Room NBW58, UMMC
22 S. Greene St
Baltimore, MD 21201