Drying at 60 degrees and other technical tips

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From:Dave Tacha <dtacha@ncal.verio.com>
To:"Histonet@Pathology.swmed.edu" <Histonet@Pathology.swmed.edu>
Date:Mon, 19 Apr 1999 08:46:38 -0700
Content-Type:text/plain; charset=us-ascii

We have done studies drying slides at 60 degrees for 1 hour, 4 hours and
24 hours.  We found that cell surface antigens such as UCHL-1 improved
with overnight heating at 60 degrees C.  However, we found a degradation
of ER staining even at 1 hour at 60 degrees C.  The longer we dried ER
slides at 60 degrees the more we saw reduced staining.  At 24 hours, ER
staining intensity was reduced by 50%.  We compared 37 degrees for 1 to
24 hours.  Staining was superior to all 60 degrees times.  We now
routinely dry at 37 degrees for 1-2 hours and then at 60 degrees for 10
minutes.  It may be even better to dry at room temperature overnight,
and then 10-20 minutes at 60 degrees C, or microwave dry for 2-4

Remember, ER and PR staining requires semi quantitative analysis.  Every
time we change one aspect of the procedure, it may change staining
quality.  This includes our drying time and temperature.  We need to
test these parameters carefully, even if we read on the Histonet that it
is okay to do!

A technique was introduced by Dr. Goldstein at the Applied
Immunohistochemistry meeting in NY.  I had the privilege to attend the
meeting last week.  Dr Goldstein presented some very interesting data on
cool down times and washing in tap water after antigen retrieval.  He
showed that a 10 minute cool down and a 5 minute wash provided the best
staining results in his lab.  If he varied from that protocol, stain
intensity and background staining changed dramatically.

Again, precise standardization is critical for all IHC procedures,
especially for ER and PR and other assays that may determine prognosis,
which in turn will influence patient treatment, and ultimately health

David Tacha HTL (ASCP)
Biocare Medical

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