RE: Proteinase K Mystery

From:"Su, Phy-Huynh" <>

I had the exact problems in 98 when I started to work with BrdU.  I used a
kit from Boerhinger Mannheim, and it worked beautifully initially.  I used
Pepsin for digestion, not Proteinase K.  Later on, when it started to give
me inconsistent results, I've changed to different antibodies from Sigma,
etc..., different antigen retrieval, etc...  Still the same problem.  Then I
realized that I've been using sections that have been cut for months, and
sitting at RT.  When I started to use only freshly cut sections, i.e.,
within 2 weeks of cutting and stored at 4 degree C, the problems went away.
I called BM and asked if they experienced the same antigenicity loss
problem, they told me that they had never used the kit on old cut sections.
Since then, I've kept all my paraffin blocks in the fridge, and only used
fresh cut sections for BrdU staining.  My senior histologist didn't want to
believe that we lost BrdU antigenicity, and he wanted me to document this by
staining sections at 1 wk, 2 wk, 3 wk, 4 wk, 3 month old cut sections, but I
didn't have time money to try that to confirm.  
Hope this would help.

> -----Original Message-----
> From:	Bennett, Catherine (Katie) []
> Sent:	Wednesday, November 15, 2000 1:35 PM
> To:	''
> Subject:	Proteinase K Mystery
> A co-worker of mine from my old lab called me the other day as she was
> having problems with a BrdU IHC procedure I had worked out before I left.
> She and I went over her problem - which was inconsistent staining results
> with some slides coming out negative, when maybe a few days earlier, they
> had come out slightly positive.  But overall, the results were never
> great.
> This is not a problem of weird background staining.  We are talking no
> staining here.
> She went back to the archives and pulled some old unstained slides from
> blocks that I had stained successfully, and still she had the same
> unsatisfactory results.  
> From what she was describing, it sounded to me that the culprit was the
> Proteinase K pre-treatment step.  There is no microwave antigen retrieval
> step to blame since we fixed with a Paraformaldehyde/Gluteraldehyde blend
> -
> and the fixation procedure has not changed for newer tissues.  There are
> no
> other pre-treatment steps except a 1N HCl denaturing step.  The staining
> uses a Vector ABC-AP kit with a Vector Red label.  (Both of which she
> bought
> new.)
> Turns out that a while back, someone in the lab switched over to buying
> the
> Proteinase K from Gibco, instead of from Sigma which is what I had always
> used.  The IHC procedure calls for a 0.01% solution, by weight.
> (Treatment
> is at 37 degrees for 30 minutes.)  So my question is, are all Proteinase
> K's
> born equal?  Could this Gibco brand of Proteinase K be weaker?  This is
> the
> only thing that seems like it is the culprit for her problems, except...
> I know antigenicity can be lost over time when unstained sections are
> stored
> for long periods of time, especially at room temperature.  These sections
> are all GMA (Immunobed).  I know BrdU is a very stable protein.  Anyone
> ever
> seen a loss of antigenicity with BrdU?  I was thinking this might be
> another
> reason why she couldn't stain the old archive slides.  But this doesn't
> explain why she would have problems with the newer cases.
> Any comments??
> *********************************
> Catherine "Katie" Bresee Bennett
> Sr. Research Technologist
> Lovelace Respiratory Research Institute
> Albuquerque, New Mexico

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