[Histonet] Tissue Washing

Tony Henwood AnthonyH <@t> chw.edu.au
Sun Aug 2 17:46:53 CDT 2009

Several ideas -

1.	Make sure the biopsies do not dry before adding to the fixative.
2.	Extend your fixation time as much as possible, decrease your
processing times (esp if you are using microwave processing)
3.	Dry the slides for 25-30 min at 64oC (60 may not be hot enough
to melt the paraffin


Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Emily
Sent: Friday, 31 July 2009 11:58 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Tissue Washing

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.

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