Re: [Histonet] Exploding skin samples
I think your skin samples are overdehydrated and a different fixation would
not improve anything. How long did you fix with NBF? If it is totally
fixed, then you will have few problems since the skin is so thin. If the
skin is NOT totally fixed the alcohol will complete the job and you will
have trouble cutting.
If the tissue is fixed completely before processing, mouse skin is thin!
should be 100% with NBF for sure.
Start in 70%, 80%, 95% X 2, 100 X 2, xylene X 2, paraffin X 2. You can keep
the same times or increase to 45 min each change.
Why are you doing alcohol xylene,, then alcohol again? Normally, one goes
to xylene changes and not back into alcohol, with the thin skin, water
carryover is probably a nil factor. Personally, I would eliminate the
xylene/alcohol, really not necessary and contributes to overdrying and
hardening of tissue, you have far more absolutes in the end than you need.
Do you NOT use vacuum and pressure? A dip and dunk situation?
Make sure your paraffin is NOT over 60C, and you could increase this to 45
min, or put another paraffin in a hot oven and have a third change to make
sure you have no xylene carryover into last paraffin.
also, keep temp of waterbath a bit cooler - not sure what paraffin you are
using nor waterbath temp.
At 05:26 PM 6/21/2004 +0100, you wrote:
>I have been given some mouse skin fixed in formalin (neutral buffered from
>Sigma, equivalent to what we used to call 10% neutral buffered formalin). I
>have tried several processes and even tried post fixing in Bouin's fluid as
>suggested by a former colleague who did a lot of rat skin sections in a
>previous job. They are spreading on the waterbath, especially around the fat
>layer at the base of the dermis. It is important to us to get good sections
>of these samples, can anyone suggest any other procedures I can try? My
>process times were 30 minutes in 50, 60, 70, 80, 90., 100% ethanol, one
>ethanol for 1 hour, 50:50 ethanol/xylene 30 mins, ethanol 30 mins, 2
>xylenes 30 mins, 2 x paraffin for 30 mins, embed. I only have 2 wax baths on
>my processor and have tried returning samples to wax for longer impregnation
>times without success.
>I propose to try out a number of different fixatives such as formol alcohol,
>methacarn, Bouin's as primary fixation and compare with NBF. Any suggestions
>would be most gratefully received.
>Thanks a lot
>University of Cambridge
>Histonet mailing list
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Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
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