Re: Water bath, fast immuno, Digest what?

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From:"Jeff Silverman" <>
Date:Mon, 21 Jun 1999 17:50:39 -0400
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Hi Netters. Let me be the first to wish everyone a happy summer (winter
down under, sorry folks, our turn now). 

Re: time constraints with immuno's. I use a 60 degree oven at all times,
place slides in immediately after cutting with minimal air dry, always use
gelatin in my water bath, and Plus (silanized) slides. Normal antibody with
no retrieval is ready in 30 minutes. Trypsin- one hour at least. Water bath
retrieval- one hour, two is better. Microwave- 2 hours to overnight.
Cellular stuff requires less time than fibrous, fatty, or suboptimally
processed blocks. In practice, most sections get overnight. I have left
toublesome blocks for ER/PR in the oven over a four day absence with great
results. Some of my stromal markers, CD34, factor XIIIa, work well after
one week in the 60 deg oven! My PBS washes are very fast, 5 sec wash bottle
rinse followed by minimum  30 seconds,usually more in a coplin jar of PBS,
which isn't changed for a ten slide run. Never a problem. Does anyone nuke
immuno antibody incubations? 

I second Dr. Cartun's remarks, the Isotemp oven from Fisher, expensive
though it may be (I have the little one- 3 coplin Jars) , works nicely for
Her2 and others, much gentler than microwave. Jules Elias and friends at
Stony Brook this month in JOH published results on enzyme digestion plus
water bath retrieval being  effective for MIB1 proliferation index.
Question- Does anyone know if Ki67 (MIB1 and other clones)  reactivity
deteriorates in stored paraffin sections? 

Nathaly from IAP, what digestion do you mean? Proteolytic digestion for
immuno, diastase for glycogen, hyaluronidase for stromal mucin? Please be
more specific. 

Jeff Silverman
Southampton Hospital NY USA

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