Re: [Histonet] Liver sections

From:Gayle Callis

Rachael,

If the animal has not been perfused with the PFA fixative, or the liver 
reduced in size, I doubt a WHOLE liver will be fixed after overnight PFA 
immersion.  We find adult murine liver dropped into NBF for a day and even=20
a few days is NOT fixed, it still looks bloody inside indicating not 
fixed.   If you "breadslice" the liver, fixation will improve since it 
surrounds all sides of the sliced tissue.

After trimming ,oversoaking is not a good idea with this very homogenous 
tissue - we trim, soak on block of ice with water on top of ice, (can be 45=20
min to 1 hour if needed) but recently improved sectioning and morphology by=20
removing the block from ice water soak but putting the block into room 
temperature or even warm water JUST before sectioning, generally improves 
sectioning.

Check the block face and if the tissue has swelled out of the block, then 
you can get thicker sections.  Make sure you back the block away from 
blade, then reapproach carefully, you do not want to cut off what you have=20
soaked, but go into the tissue just a bit away from the tissue  - you do 
not want to lose the advantage of the soak.

Also, readdress your processing, you may be exposing the liver to solvents=20
i.e alcohols way too long aka overprocessing.  This removes not only the 
free water from tissue spaces (that is what you want) but also the bound 
water on the protein (you do NOT want this to happen) which turns liver 
(also spleen) into hard little nuggets that shred during 
sectioning.   Soaking helps, but it is still an annoying problem.  Poor 
infiltration of paraffin can also contribute to shredding.

At 05:47 AM 5/1/2006, you wrote:
>Hello. I need some advice about cutting adult mouse livers. The livers are
>fixed in paraformaldehyde overnight, rinsed, stored in 70% EtOH, and then
>processed into paraffin.  I usually cut at about 4um, but my sections look
>like they are thicker...maybe 10um.  Everything else I cut looks fine, it=B9s
>just the liver.   Usually I face the block and let it sit on ice for 1-2
>hours before cutting.  Sometimes if I am having trouble with shredding, I
>will let it sit overnight on ice at 4C.  Any advice or suggestions on my
>thickness problem would be greatly appreciated.
>
>Thank you
>Rachael
>
>--
>Rachael L. Emerson
>Center for Pediatric Biomedical Research
>University of Rochester Medical Center
>575 Elmwood Avenue MRBX 1.11301
>Rochester, NY 14642
>
>
>
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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
406 994-4303 (FAX)



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