RE: tissue processing lets do mouse spleen

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From:"Joyce Kotzuk" <> (by way of histonet)
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Thank you for some important information. My last batch of mice came out a
little dry, and I'm thinking 1) I let the temperature of the paraffin get
too high (putting 20 cassettes into the wax caused it to start to harden,
so I turned up the oven; when I checked it, the temperature was nearly 70
degrees C) or 2) they were too long in 100% EtOH/Cedarwood oil (50-50 mix).
Although the cedarwood oil is protective, the 100% EtOH may still have been
too drying. I do 3x15 minutes in 100% EtOH/Cedarwood oil. I don't use
xylene at all, cedarwood oil and methyl salicylate instead, then paraffin.
Comments/questions about this are welcome.
Joyce Kotzuk, Univ of New Mexico pathology dept.

>>> "Sarah Christo" <> 11/18/99 01:38PM >>>
Dear Everyone having problems with mouse tissues.  I am the guru of mouse
tissues.  I live, eat and breath mouse tissues.  I can process, embed cut
stain mouse tissues in my sleep.
  First, it has to be properly fixed.  If it ain't fixed right don't
bother.  Your fooling around with your processing schedules, clearing
agents and standing on your heads and spitting nickels will not help.  You
can not trouble shoot improperly fixed tissue.  If you are not perfusing
mouse tissues you are not optimizing fixation.
  Second.  If you have your happy perfused mouse and dissect out your happy
meeses pieces, start with a 15 minute processing schedule.  Vacuum and
pressure or fancy do dads on your processor are not necessary.  They can be
processed by hand if you know what you are doing.
  Third.  Try to section it.  If you are having to soak the little suckers
for hours, cut back on your processing time until you have a block that
only has to soak 5-10 minutes.  I haven't seen a mouse spleen or liver that
does not have to be soaked for at least a short time.  They just are
naturally crispy critters.
  Fourth.  If you are using xylene on the processor it will be more crispy.
It is still OK to use xylene, I do, but consider that in your thinking of
times in the stations.
  Fifth. Separate out the different sizes of tissues.  The spleen cut in
cross section is very small.  Fumes would probably process it.
  OK, I'm off my soap box.   Have a Happy
I think I need to go home and pet the dog or something.....

Sarah Christo, HT (ASCP)
Texas A&M University
College of Veterinary Medicine
Dept. of Vet. Anatomy & Public Health
Histology Laboratory
College Station, TX  77868-4458
phone (409) 845-3177
fax (409) 847-8981

>>> Cynthia Favara <> 11/18 11:30 AM >>>
	Just a question in this regard. I attended a workshop about 4 years
ago and the information given was that specimens could be over dehydrated,
and that is why it is necessary to soak blocks. Now what I am apparently
hearing is that it is not enough dehydration [ I was going to say
underdehydration but don't know if that is a correct term] and this doesnot
allow for adequate penetration of the remaining reagents. So if this is
correct when a block has to be soaked inorder to be cut it is because the
specimen has not been adequately dehydrated and increasing the processing
time should help??? Have I got it right. Please let me know I would love to
be able to do rodent spleen and liver without having to soak.
Thanks,Cynthia Favara
903 South 4th Street
Hamilton, MT 59840
PH: 406-363-9317
FAX: 406-363-9286

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