Catherine wrote:

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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Dear Catherine,

Indeed antigenicity can be lost in stored paraffin slides. There is even
some literature available on this subject (see below). I have encountered
the same problem with the Ki67 antigen, which happens to be also an nuclear
antigen.

How about actual comparison of the two types Proteinase K?

Furthermore, BrdU can be stained very well without all those funny
pretreatments (HCl and Proteinase K) replacing them by simple antigen
retrieval in citrate buffer pH 6.0 or Tris-HCl/EDTA pH 9.0.

Good luck, Chris




Jacobs TM, Prioleau JE, Stillman IE, Schnitt SJ: Loss of tumor
 	marker-immunostaining intensity on stored paraffin slides of breast
 	cancer. J Natl Canc Inst 1996; 88:1054-9
Kato J, Sakamaki S, Niitsu Y: Re: More on p53 antigen loss in stored paraffin
	slides. N Eng J Med 1995; 333:1507
Manne U, Myers RB, Srivastava S, Grizzle WE: Re:loss of tumor
	marker-immunostaining intensity on stored paraffin slides of breast
	cancer. J Natl Canc Inst 1997; 89:585-6
Prioleau JE, Schnitt SJ: p53 antigen loss in stored paraffin slides. N Engl J
	Med 1995a; 332:1521-2
Prioleau JE, Schnitt SJ: Re: Re: p53 antigen loss in stored paraffin slides.
	N Engl J Med 1995b; 333:1507-8
Songun I, Van der Velde CJH, Kramer-Knijnenburg G, Van Krieken JHIM:
	Detectability of retinoblastoma (Rb) gene product and p53 antigen
	expression decreases in stored paraffin sections. Histopathol 1997;
 	30:604-7