RE: Disappearing Cytoplasm!!

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From:"margaret blount" <>
To:"Bruce Abaloz" <>, "" <>
Date:Tue, 21 Sep 1999 09:20:59 +0100

I would maintain the sedtions in PBS, even after NBF fixation, they won't be
very well-fixed and you may be causing osmotic damage by transferring them to
distilled water. I don't believe that dehydration is removing  the cytoplasm.
When you pick up your sections are you touching the knife? This will cause
artefacts due to the tissue adhering to the knife and leave the tissue looking
Hope this helps.
Unilever Research

-----Original Message-----
From:	Bruce Abaloz []
Sent:	Monday, September 20, 1999 8:22 AM
Subject:	Disappearing Cytoplasm!!

Hi all....I'm a student here @ the Uni.of Melbourne & am freezing fresh liver &
spleen (mouse), floating the OCT covered specimen in a boat in liquid nitrogen
until OCT is a solid block then I drop it all into the liquid nitrogen. I store
it @ -70C until ready to cut in the cryostat.I am using Histogrip slides & cut
@ 6 microns @ -10C.I let it dry @ room temp.for 1/2 hr.- 2 hrs.then fix in 10%
NBF for 10 min. rinsing X3 in PBS. after which I transfer to DH2O to transport
to the Histology Lab. for H&E staining. I have no problem with the stain but
the sections after staining, look as if the cytoplasm has been "dehydrated
anyone advise me on what "to do" or what I'm doing wrong. I've also tried
Acetone fixation (10 min.@ RT) with the same result??????????? I look forward
to your replies & THANKYOU in advance.........Simone Cuff.


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