[Histonet] Exploding skin samples

[Margaret Blount]

Dear Gayle,

Sorry for the tardy reply, I have been rather busy. Thank you for your
valuable advice, I have learnt to respect you as a professional just reading
your postings.

The skin was in NBF initially for 2 days and by the time I tried the latest
and rather eccentric method it was about 10 days! I was given a 1cm x 1cm
square or thereabouts. This process was a last resort after other techniques
didn't work and I must admit that I haven't handled rodent skin since the
1980's so I'm rusty! I had seen postings about fatty tissue and tried this
in the hope that it might work. I do wonder what the researchers are doing
with the skin prior to putting it into NBF, maybe it is lying around a
while?

My processor is a Leica dunk and dip TP1020 with vacuum. I can certainly use
your suggested process on it. My original process which I acquired in my
last lab is much longer and I tried it without success, I won't bore you
with the details as I came to the conclusion it was too long. It does work
very nicely for some tissues I have to say.

The process I described to you was done by hand except that I used the wax
stations on the processor to have the benefit of vacuum. 

i originally floated out at about 46 and soon realised I had to lower the
temperature, taking it down to 36 before deciding to ask my question on the
histonet. I have been told to lower it even further, even as low as room
temperature and then raising it untilI get a satisfactory result. Also the
suggestion of adding alcohol to the waterbath has been raised, though no one
said how much. On that subject, I was trained originally to float my
sections on to 20% ethanol prior to floating them on the waterbath (I think
that's too strong and use 15%) and would normally do this for tissues that
cut well, so I can see the value of adding alcohol to the waterbath. However
I am afraid that tissue that tends to spread might be worse with the
alcohol, certainly I don't use alcohol in my usual way for such sections.

I think I have covered everything and I'm in danger of rabbiting on! 

Thank you for your help. I appreciate it.

Regards

Margaret



-----Original Message-----
From: Gayle Callis [mailto:gcallis <@t> montana.edu]
Sent: Monday, June 21, 2004 6:29 PM
To: Margaret Blount; Histonet <@t> lists.utsouthwestern.edu
Subject: 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
>
>Margaret
>
>Margaret Blount
>Chief Technician
>Clinical Biochemistry
>University of Cambridge
>Addenbrooke's Hospital
>Hills Road
>Cambridge
>CB2 2QR 
>
>
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>
Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)