Re: [Histonet] eosinophils on frozen section

From:"Gayle Callis"

We improved our eosinophil staining on frozen sections of murine tissue by 
switching to NBF immersion fixation, let it sit 10 min, then do the H&E but 
use eosin/phloxine (Richard Allan stain will work) but stain in 
eosin/phloxine for 2 minutes.    For some reason, the eosin was never taken 
up as well after a shorter time in eosin alone,  and greatly improved with 
the phloxine added.  Sections can be cut a bit thinner too, try 4 um - it 
should make the granules more apparent against the background and other 
cells. Also, I suggest adding absolute alcohol after the 95% for better 
dehydration before clearing.

You may have success using alcoholic formalin in order to speed up the 
fixation a bit.  However, when we switched to NBF, we had better staining of 
eosinophils in frozen sections.  If you have the time, you can let NBF 
fixation go longer, we often let sections sit for days then stain, but with 
your diagnostic procedure, you will want a bit more speed involved.

Good luck

Gayle M..Callis
Bozeman MT 59715

----- Original Message ----- 
From: "Breisch, Eric" 
Sent: Tuesday, February 05, 2008 4:32 PM
Subject: [Histonet] eosinophils on frozen section

To all interested Histonetters:

We are experiencing some frustration with our ability to identify
eosinophils from frozen sections. For some reason which our frozen
section set up does not enable us to identify eosinophils. Eosinophil
identification is not impaired when the tissue is submitted for
permanents. Could some one please share the reagents used and how the
frozen section staining area is set up if they have success identifying
eosinophils from a frozen section? We have had many frozen sections done
on fresh tissues suspicious for an eosinophilic granuloma but our
techniques so far have not rendered consistent eosinophil
identification. We presently are using 95% ETOH for fixing the FS slide,
quick wash in distilled H2O, 1 minute in Richard Allen Hematoxylin 2,
rinse in tap water, 15 seconds in bluing, rinse in water, rinse in 70%
ETOH, 1 minute in Richard Allen Eosin Y, three rinses in 95% ETOH, three
rinses in Xylene substitute and then coverslip. All other tissues appear
just fine but the eosinophils just don't show up. Any suggestions are
greatly appreciated.

Eric A. Breisch, Ph.D.

Clinical Anatomist

Dept. of Pathology

Rady Children's Hospital and Health Center

Associate Clinical Professor of Anatomy

Dept. of Surgery

UCSD School of Medicine

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