Re: mouse ovaries

From:Andrea Grantham

Sometimes I think I have become the queen of mouse ovaries here. The ovary 
people are my best customers! The ovaries are fixed in 10% NBF for a short 
time then transported to the lab in 70% ETOH. For some of the projects that 
they do they fix in Bouins and transfer to 70% ETOH before they come to the 
lab. I often have to wash out the Bouins before processing and this is real 
important because old blocks that were done before my time are still full 
of Bouins and cut terrible - very brittle.
I use an old processor that doesn't have any of the fancy heat and vacuum 
functions and it uses just a gentle agitation. I process for one hour in 
each station starting in 70% ETOH x2, 80% ETOH, 95% ETOH x2, 100% ETOH x3, 
ClearRite 3 x2, and Paraffin x2 (currently I am using Richard Allan Type 6 
but I like the Surgipath Formula R as well). I cut serials on many of the 
ovaries or step sections and they cut like a dream. I also receive cultured 
ovaries that are so small that they can barely be seen and larger rat 
ovaries and use the same schedule and have no problems with them. I cut the 
blocks at room temp and only on a rare occasion have to soak the blocks 
before sectioning. We get beautiful H&E staining and IHC.

At 10:03 AM 6/3/2003 -0600, you wrote:
>Shorten your processing schedule - overdehydration, over clearing in xylene
>(we now use Clearite 3 for all tissues, including decalcified bone) and
>overexposure to heat of paraffin will cause dry, friable tissues.  It also
>helps to face block, soak on ice block with water. You did not say what
>kind of processor you have, but adding heat to processing through solvents
>also is drying to murine tissue.
>You may want to try
>  30 min in 80% ethanol (skip the 70% ethanol(?), you have already done that
>  2 changes 95% ethanol at 30 min per change
>  2 changes 100% ethanol at 30 min per change
>  2 changes Clearite 3 at 30 - 45 min per change (a good alternative is 1
>change xylene, then 1 change            Clearite 3 - this seems to help on
>crunchieness issue)
>  2 changes paraffin (try to use lower melting point i.e. 56C) 30 minutes
>each change, a third change can be use.
>If you have a VIP or other automatic processor, program it so the total
>time in 2 changes of 95% is spread out over three changes  (i.e. 15, 15, 30
>If the tissue was fixed in 70%, it may be crunchier to begin with,
>hopefully NBF was used prior to storage in 70%.
>30 At 10:28 AM 6/3/2003 -0500, you wrote:
> >        A Histonet search revealed that you have been working with mouse
> >tissue for a long time.  We have done mouse ovaries before but they have
> >turned out crunchy.  I was wondering what you would suggest for a
> >processing schedule.  The tissue will be in 70% alcohol.  We do not know
> >any more details on fixation of the tissue.       Thank you for your time.
> >Margaret Perry  Lab Tech Histopath  South Dakota State University
> >Brookings SD 57007  605-688-5638
>Gayle Callis
>Research Histopathology Supervisor
>Veterinary Molecular Biology - Marsh Lab
>Montana State University - Bozeman
>S. 19th and Lincoln St
>Bozeman MT 59717-3610
>406 994-6367 (lab with voice mail)
>406 994-4303 (FAX)

: Andrea Grantham, HT(ASCP)     Dept. of Cell Biology & Anatomy     :
: Sr. Research Specialist       University of Arizona               :
: (office:  AHSC 4212)          P.O. Box 245044                     :
: (voice:  520-626-4415)        Tucson, AZ  85724-5044    USA       :
: (FAX:  520-626-2097)          (email:       :


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