Re: Antigen Retrieval

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From:Amos Brooks <> (by way of histonet)
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    Proteinase K (PK) is an enzyme digestion meant to take the place of heat
induced epitope retrieval (HIER). For many antibodies this works great. But you
shouldn't do both. Try the Vimentin using HIER and no PK or vice versa.
it may be that you may need to use HIER on one and PK on the other both do not
need to be run the same since they are different antibodies.
Amos Brooks

"Bennett, Catherine (Katie)" wrote:

> 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
> BrdU.
> Anyone have any insight or suggestions?
> ****************************
> Catherine "Katie" Bresee Bennett
> Sr. Technical Associate
> Lovelace Respiratory Research Institute
> Albuquerque, New Mexico

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