Re: Mouse tissue processing protocol

From:

Bryan Llewellyn

RE: Mouse tissue processing protocol

I too am of the opinion that overdehydration and overprocessing are myths. Usually this artifact arises with small biopsies (often gut) which have been given minimal fixation in NBF, followed by rapid processing in a modern processor with full heat in the dehydration step. I believe that the effects beeing seen are due to the tissue getting insufficient (or even no) chemical alteration from the formalin because the time is too short. This is then followed by lousy fiixation in hot ethanol under the guise of dehydration. Check out the old texts to see what ethanol fixation results in, then exaggerate it, and you will get a description of this artifact. Perhaps the resolution is to develop a rapid fixative just for this purpose, but which protects the tissue against ethanol fixation.

Bryan Llewellyn

----- Original Message -----

From: Hewlett Bryan

To: histonet@pathology.swmed.edu ; 'Renee Hoyle-Thacker'

Sent: Thursday, January 17, 2002 5:36 AM

Subject: RE: Mouse tissue processing protocol

Renee,

At the risk of inciting the wrath of my histology colleagues, I have to tell you that,
within any reasonable time frame( say 72 hours per change of reagent),
there is no such thing as 'over-processing'. This should be regarded as an urban myth!
As is 'over-dehydration'. It simply does not happen.
As evidence, I invite you to examine the work produced in the days before
automated tissue processors. Depending upon the tissue, between 4 and 21 days of
processing were not uncommon. The first two years of my professional life were spent
hand processing(3 day process) over 30,000 mouse and rat tissue blocks for pharmaceutical
research. Never saw the effects you ascribe to 'over-processing'.
Several times, in the past 10 years, we have had to perform blinded studies, comparing
tissues processed by both short and very long schedules, in order to convince the doubting.
Long schedules always win hands down!
Assuming that the tissue is optimally fixed, your problem is most likely insufficient time in processing reagents or a poorly designed processing schedule. We process mouse tissue that has been fixed in NBF for a minimum of 24 hours, then transferred to 70% ethanol and stored for up to 3 days prior to an overnight(16 hr) schedule with both solvent heat(37-45C) and alternating pressure/vacuum.

Regards

Bryan

----------
From: Renee Hoyle-Thacker[SMTP:rthacker@ciit.org]
Sent: January 16, 2002 2:46 PM
To: histonet@pathology.swmed.edu
Subject: Mouse tissue processing protocol

Hello everyone,

I was wondering if research people would be willing to share their
processing schedule particularly their mouse tissue protocol. We have had
trouble with dry liver, spleen, sternum, etc. At times we must soak blocks
for 1 hr or more and still obtain dry sections, we think the problem might
be over-processing. Thanks for your help!

Renee

Renee Hoyle-Thacker
CIIT Centers for Health Research
6 Davis Dr.
Research Triangle Park NC 27709
Voice: (919) 558-1322

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