Proteinase K Mystery
From: | "Bennett, Catherine (Katie)" <cbennett@lrri.org> |
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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