[Histonet] Tissue Washing

[Rene J Buesa]

Emily:
I don't see any problems with your staining protocol. I think that the problem still resides in the processing and that you are probably having a too long dehydrating period. Also, depending on your clearing agent, that dehydrating problem can be exacerbated.Try to reduce your dehydrating steps (for small biopsies) and check the characteristics of your clearing agent.
René J.

--- On Fri, 7/31/09, Emily Butte <emily <@t> pathlabsolutions.com> wrote:


From: Emily Butte <emily <@t> pathlabsolutions.com>
Subject: [Histonet] Tissue Washing
To: histonet <@t> lists.utsouthwestern.edu
Date: Friday, July 31, 2009, 9:57 AM


Our lab has been struggling for a while now, with tissue washing off the
slides. The problem was originally traced to our processor that was
malfunctioning and having major carry-over issues, causing poor tissue
processing. So after failed attempts to get the old machine fixed, we ended
up getting a new microwave processor. The tissue (mainly prostate bx) comes
out of the processor looking good. It cuts well, but after staining, some of
the sections are no longer on the slides. The problem was quiet severe with
the old processor and has decreased drastically, but has not entirely gone
away with the microwave processor.

Here's some info on our current procedure. This is a small physician office
lab and we everything manual. We use plus slides and a water bath with no
tissue adhesive added. The bath is between 42-45 degrees Celsius. After
sectioning, the slide are put in an oven at 60 degrees for 20 minutes. After
taking the slides out of the oven, we cool them to room temp then hand stain
them in an H&E line.
Xylene - 5 min
Xylene - 2 min
Xylene - 2 min
100% Alcohol - 30 sec
100% Alcohol - 30 sec
100% Alcohol - 30 sec
95% Alcohol - 30 sec
Rinse in DiH2O
Hematoxylin - 2 min
Rinse in DiH2O
0.50% Acid Alcohol - 2 dips
Rinse in DiH2O
Scott's Tap Water - 10 sec
Rinse in DiH2O
95% Alcohol - 30 sec
Eosin - 30 sec
95% Alcohol - 30 sec
100% Alcohol - 30 secs
100% Alcohol - 30 secs
100% Alcohol - 30 secs
Xylene - 30 sec
Xylene 2 min
Xylene - 2 min

The slides are then cover slipped and of a 12 piece prostate case, at least
a section or 2 will wash off of about 4 of the slides. Please pass along any
ideas as to what the cause of this could be and how I can fix it. I'm fresh
out of ideas and would really appreciate the advice of someone with more
experience than myself.

Thanks,
-- 
Emily
Path Lab Solutions Inc.
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