Re: Antigen Retrieval

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From:"Joyce Kotzuk" <> (by way of histonet)
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Can you do the vimentin first, then do HIER and the BrdU staining second?
I don't know if it would work, if the HIER would somehow ruin the vimentin
staining, but it may be worth a try.
Joyce Kotzuk, Univ. of New Mexico pathology dept.

>>> "Bennett, Catherine (Katie)" <> 12/23/99 09:51AM >>>
I realize not many histonetters may be online right now, but to those out
there, I present my current dilemma.

I am trying to do double immuno staining for BrdU and Vimentin in 10%
neutral buffered formalin-fixed rat lung sections.  I can get great BrdU
labeling with almost all of the microwave antigen retrieval procedures I've
been using (1% Zinc Sulfate, 0.01M Citrate Buffer, 0.1MTris-HCl, and 1mM
EDTA).  But the Vimentin stain after microwave AR is really weak.  Without
antigen retrieval, I get no BrdU staining but awesome Vimentin stains.  My
protocol always includes a 5 minute 0.05% Protease K enzyme digestion step,
and a 1h HCL denaturation step.  Without the microwave AR, these alone won't
do the trick for the BrdU.

I'm guessing my next step is to ditch the microwave AR since it kills the
vimentin and play around with various enzyme digestion times to boost the

Anyone have any insight or suggestions?

Catherine "Katie" Bresee Bennett
Sr. Technical Associate
Lovelace Respiratory Research Institute
Albuquerque, New Mexico

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