[Histonet] Exploding skin samples

Tue Jun 22 11:41:58 CDT 2004

If you use extended paraffin in an extra last step for better infiltration,
use a vacuum oven that heats and draw a vacuum to 20 psi.  Make sure the
temperature is not exceeding what you use on processor.  A beaker of melted
paraffin can be kept in one of these ovens.  A vacuum oven has saved us
more than once for extra infiltration.

At 08:25 AM 6/22/2004 +0100, you wrote:
>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
>
>Margaret Blount
>Chief Technician
>Clinical Biochemistry
>University of Cambridge
>Addenbrooke's Hospital
>Hills Road
>Cambridge
>CB2 2QR 
>
>-----Original Message-----
>From: Vinnie Della Speranza [mailto:dellav <@t> musc.edu]
>Sent: Monday, June 21, 2004 8:19 PM
>To: froyer <@t> bitstream.net; Histonet <@t> lists.utsouthwestern.edu
>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" <froyer <@t> bitstream.net> 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).
>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 
>>>
>>>
>>>_______________________________________________
>>>Histonet mailing list
>>>Histonet <@t> lists.utsouthwestern.edu 
>>>http://lists.utsouthwestern.edu/mailman/listinfo/histonet 
>>>
>>>
>>>    
>>>
>>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)
>>
>>
>>
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>>
>>  
>>
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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)