Processing Carnoy's Fixed Tissue

From:Gayle Callis

After Carnoys fixation (1 hour to overnight. If you tissue is not totally
fixed after 1 hour, alcohol in processing will probably complete fixation
for you unless your tissues are biospy size, extremely small. 

After fixation, tissues should be rinsed with 95% or 100% ethanol to get
rid of chloroform and acetic acid. At this point your tissues are basically
dehydrated!  You can skip all dehydration steps of processing up through
95% and go directly to 100% alcohol steps, clearing and paraffin
infiltration. We never go back to 70% or any lesser alcohol concentrations
- no need to rehydrate before processing.  That is one of the joys of
Carnoys - and it shortens the processing schedule.  

Usually, a good general histotechnic textbooks tells you how to handle
Carnoys fixed tissues - Theory and Practice of Histological Techniques, by
Bancroft and Gamble, Fifth Edition, Churchill Livingston, London has an
excellent discussion on Carnoys.  They recommend up to 5 hours fixation for
small biopsies and after 1 hour fixation, tissues should be transferred
directly to 100% ( I still recommend a 95% or 100% alcohol RINSE to avoid
acid contamination of your processing alcohols at this step).

 At 09:23 AM 9/18/02 +0100, you wrote:
>I'm emailing from Manchester in the UK and just starting my histology 
>career.  Please can anyone help?
>Just read some archived mail on Histonet regarding processing carnoy's 
>fixed tissue - I really need some help with this, as we are getting strange 
>black/brown nuclei throughout the tissue.  It has been suggested that this 
>is the acid remaining in the nuclei.  We have a Leica carousel tissue 
>processor without a vacuum, however, I have used an overnight 18 hours 
>program and still can't get rid of this. We also use other fixatives which 
>aren't a problem, but we would like to resolve the problem with carnoy's 
>fixed tissue, as we use this fixative a lot in our lab and it is necessary 
>for soem of our work.  Tissues are fixed for approx. 1 hour in Carnoy's and 
>then transferred to 70% prior to processing.
>I have also read on Histonet that there are 2 references regarding the 
>processing of carnoy's fixed tissue, however, there aren't any abstracts on 
>PubMed and I'm not sure whether our library holds this journal.  Does 
>anyone know how I can get hold of them?  They are:-
>Puchtler H et al  Histochemie 16:361-371, 1968
>Puchtler H et al  Histochemie 21:97-116,	1970
>If anyone has any other advice for dealing with these tissues, I would be 
>very grateful if you could pass it on.
>Kind regards,
>Nicola Cragg
>Epistem Ltd
>Manchester, UK
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


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