RE: [Histonet] Exploding skin samples

From:Margaret Blount

Dear Vinnie,

Thanks for your reply. My adding an ethanol after xylene was an experiment
after I read about fatty samples not processing well and several authors
seem to do this and I hoped it would help. It hasn't but I take your point
about the impregnation with wax possibly being insufficient. I tried
returning earlier samples to wax without success and I have obviously got
try again. Maybe combining this with the other suggestions of lowering the
waterbath temperatures even more.

Thanks again


Margaret Blount
Chief Technician
Clinical Biochemistry
University of Cambridge
Addenbrooke's Hospital
Hills Road
CB2 2QR 

-----Original Message-----
From: Vinnie Della Speranza []
Sent: Monday, June 21, 2004 8:19 PM
Subject: Re: [Histonet] Exploding skin samples

I'm inclined to agree with Ford. exploding sections have not been adequately
infiltrated with paraffin. This could be due to water or fat that was not
effectively removed during dehydration and clearing. 

One further note. On a processor that only has two paraffin stations, I
encourage you to reduce the time in the first paraffin as it is almost
always contaminated with xylene. the second paraffin is the important one in
this scenario so you might consider using 15 or 20 minutes in the first
paraffin and maybe 45 minutes in the second.

Lastly, Gayle is correct that alcohol should not follow xylene in your
processing scheme. Your tissues must be infiltrated with xylene if you are
to succeed in paraffin infiltration.

Vinnie Della Speranza
Manager for Anatomic Pathology Services
Medical University of South Carolina
165 Ashley Avenue  Suite 309
Charleston, SC 29425
Ph: 843-792-6353
fax: 843-792-8974

>>> "Ford Royer"  06/21/04 02:11PM >>>
It has been a loooong time since I did this, but what sticks in my mind 
is that tissue sections would "explode" in the water bath if they had 
not be dehydrated enough. i.e. Water was still present in the 
intracellular structures either from the original cytoplasm (tissue not 
completely fixed) or from the NBF.  Again if memory serves, we increased 
the times for the alcohol to make sure all of the water was out of the 
cells before bridging through xylene to paraffin.

....or do I have this all backwards?

~ Ford
Ford M. Royer, MT(ASC)
Former Lab Rat
Analytical Instruments, llc
Minneapolis, MN 55427
(800) 565-1895, Ext. 17

Gayle Callis wrote:

>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).
>>have tried several processes and even tried post fixing in Bouin's fluid
>>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
>>layer at the base of the dermis. It is important to us to get good
>>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
>>my processor and have tried returning samples to wax for longer
>>times without success. 
>>I propose to try out a number of different fixatives such as formol
>>methacarn, Bouin's as primary fixation and compare with NBF. Any
>>would be most gratefully received.
>>Thanks a lot
>>Margaret Blount
>>Chief Technician
>>Clinical Biochemistry
>>University of Cambridge
>>Addenbrooke's Hospital
>>Hills Road
>>CB2 2QR 
>>Histonet mailing list
>Gayle Callis
>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)
>Histonet mailing list
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