[Histonet] Re: Processing mouse pancreas

[gayle callis]

It was written:  
 
Develop a good general protocol for animal tissue. You cannot have
"dedicated" protocols for every type of tissue.
René J.
 
--- On Fri, 6/3/11, Stoll, Kathryn <kstoll
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t> mcw.edu>
wrote:
 
From: Stoll, Kathryn <kstoll
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t> mcw.edu>
Subject: [Histonet] Processing Mouse Pancreas
To: "histonet  <http://lists.utsouthwestern.edu/mailman/listinfo/histonet>
<@t> lists.utsouthwestern.edu" <histonet
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t>
lists.utsouthwestern.edu>
Date: Friday, June 3, 2011, 12:03 PM
 
Happy Friday Everyone,
Does anyone have a protocol for processing mouse pancreas?  We have a VIP6.
I am not familiar with the processing any animal tissue and do not know if
it is similar  to human.
 
Thanks in advance.
 
Kathryn Stoll, HT(ASCP)
Depatment of Pathology
Medical College of Wisconsin
9200 W Wisconsin Ave
Milwaukee WI 53226
414.805.1525
kstoll  <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t>
mcw.edu
 
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Sorry, but I couldn't disagree more about developing an overal(?)l  general
protocol for animal tissue.   This may work for diagnostic veterinary
laboratories where daily runs require a single overnight schedule, but in a
research laboratories where one works almost exclusively with the mouse or
other rodent  animal models,  then different or custom processing schedules
are common.  This is the case with our research laboratory.   We avoid "over
processing" or over exposure to solvents, effects of clearing agents and
paraffin heat as much as possible and find different schedules work better
depending on size and type of tissue being processed.  

 

Mouse pancreas is very small, so avoid "over processing" e.g. lengthy
processing in each solvent, clearing agent and hot paraffin to not dry out
the tissue resulting is difficult sectioning.   For pancreas or other small,
delicate murine tissues, I suggest you use a short schedule.  This is much
like a biopsy schedule found in clinical laboratories.  In fact, you can use
your biopsy schedule  or at least compare it to the following short
schedule.   We have a VIP6, an  extremely efficient machine compared to our
old VIP, and had to readjust/shorten the processing times for spleen, liver,
single lung lobes, lymph nodes, spinal cords cord cross sections, tiny
embryos,  etc.  Some researchers have as many as three to four different
schedules depending on what organs they are processing.    One researcher
has three custom schedules for brain and spinal cord depending on whether
the brain is whole, sliced or spinal cord is in cross sections.   For whole
lungs, or larger tissues, we use a longer schedule probably similar to your
routine clinical schedule, and for bone, much longer processing due to
density of the decalcified bone matrix. 

 

We have used the following short schedule with success, and this should work
for the pancreas.   

 

Tissue should be totally fixed before putting on the processor since all
processing starts 70% ethanol.  If formalin is on the machine, then one can
use the processor setup which will probably have two changes of 95% and 100%
alcohols, and increase the times accordingly to ensure good dehydration.  We
use pressure and vacuum, no heat added to any solvents (heat lends to more
drying of lean animal tissue).   

 

70% ETOH                                            15 minutes

80% ETOH                                            15 minutes

95% ETOH            (3 changes)         15 minutes each  - if two stations,
use 25 minutes per change

100% ETOH         (3 changes)        15 minutes each  -  if two stations,
use  25 minutes per change

Xylene                  (1 change)           15 - 20 minutes

Clearite 3             (1 change)           15 - 20 minutes

Paraffin at 60°C  (3 changes)       20 minutes per change.   

 

You can use two xylene changes if you already have that on your VIP6.   You
can also use Clearite 3, Propar  or another xylene substitute exclusively
but we avoid limonene based xylene substitutes (sensitizing and makes people
sick due annoying orange citrus odor), but increase the time (20 - 25
minutes)  per change in the substitutes. These are more sensitive to
residual water carry over.  We like to ensure the alcohols with any residual
traces of water are cleared well with xylene in first step, and then finish
with Clearite 3 in second step.   Clearite 3 or Propar doesn't harden the
tissue excessively making sectioning easier.  Paraffin choice is whatever
the lab prefers.   IF you find you need longer processing, increase the time
by 5 minutes in each change including the paraffin.  

 

Good luck

 

Gayle M. Callis 

HTL/HT/MT(ASCP)

GCallis Histology Service LLC

Bozeman MT