[Histonet] RE: IHC Doublestain

[Piero Nelva]

Hi Kimberly

I think that this is a classic "try it and see" problem :-), but HEIR before 
enzyme sounds best.  I've combined dual staining, but only with the same 
pretreatment.  It may be a case of compromising on the staining intensity 
for each antibody, rather thatn optimising either.

THat said, I always like to see dual staining.  Sometimes there is just too 
much DAB brown and a splash of any other colour brightens my IHC day.

Regards

Piero Nelva
Anatomical PAthology
Monash Medical Centre
----- Original Message ----- 
From: "C.M. van der Loos" <c.m.vanderloos <@t> amc.uva.nl>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Thursday, January 08, 2009 8:03 PM
Subject: [Histonet] RE: IHC Doublestain


> 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
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