[Histonet] RE: Double staining of 2 antibodies with 2 different
With this type of trouble there are usually no solutions. However, in
your case there is perhaps one:
You may perform your double staining sequentially starting without
antigen retrieval. Incubate with the ubiquitin antibody followed by an
appropriate polymer/HRP. Visualize HRP activity with DAB
(obligatory!). Then perform HIER with citrate and continue with the
HLA-DR antibody etc. ending with AP activity in red (allowing a weak
hematoxilin counterstain). In general, a brown-red color combination
is not suitable to observe real co-localization by mixed colors.
However, you said that you are looking for structures near to each
other, so this will be no problem for you.
Hope this helps!
Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
NL-1105 AZ Amsterdam
Date: Fri, 19 Jan 2007 11:20:44 -0800
From: "Patrick Laurie"
Subject: [Histonet] Double staining of 2 antibodies with 2 different
I am trying to do some double labeling of ubiquitin and HLA-DR LN3
antibody on human spinal cord. My PI wants to be able to see both the
ubiquitinization of the motor neurons with patients that had ALS, as
well as seeing the proliferation and activation of microglia on the
slide, near the same cell. But, I have found that the ubiquitin I am
using, (Chemicon's Monoclonal ubiquitin antibody) requires no antigen
retrieval (AR), while the HLA-DR LN3 antibody (monoclonal from MP
biomedical) works best with a 30 min citrate retrieval. The citrate
retrieval with the Ubiquitin creates too much background staining,
blocking reagent! s are pow
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