RE: ER/PR on cytological smears

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From:"Sebree Linda A." <> (by way of histonet)
To:histonet <>
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Hi Beverly,

Wish I had some good suggestions for you.  I spent considerable time trying
to get ER/PR to work on cytology preparations.  I did have one protocol
work, once, but was unable to reproduce it.  These were all on cytospins and
ThinPreps prepared from ER/PR positive cell cultures.  The one time I
achieved appropriate staining was on ThinPreps (fixation protocol for
preparing ThinPreps is: fixation of cell culture cells for 15" in
PreservCyte which contains 50% methanol, ThinPrep procedure followed by 15"
in 95% ETOH followed by air-drying).  Slides were stored at -20 degrees C
until needed and brought to R.T.  prior to staining.  I then post-fixed them
in Ventana's Morphosave which is a sodium bisulfite reagent designed to
preserve morphology of frozen sections, cytology preparations and blood
smears.  They were then treated to 5" of HIER in 1mM EDTA, pH 8 at 91-93
degreees C and then allowed to cool for 20".  These were then stained on a
Ventana immunostainer using Ventana antibodies: ER (6F11) and PR (1A6) for
32" incubatioin.  Avidin Biotin block was applied as well as the Ventana
Amplification kit (RxM IgG and MxR IgG) after the primary.  Slides were
lightly counterstained with hematoxylin.

This was the only protocol that resulted in appropriate nuclear staining and
clean cytoplasm.  Everything else I tried yielded cytoplasmic staining
without nuclear staining (or cytoplasm and nuclei exhibiting the same degree
of staining.)  Unfortunately, as I said, I was unable to reproduce it.

I will be interested in seeing if any other histonetters respond with
protocols that yield the desired results.  Please let me know if you achieve
success.  Our whole mission to this project was to have a ready supply of
ER/PR controls for cytology preparations. But as it turns out we're back to
using frozen sections as the next best alternative (optimizing frozens for
ER/PR is an entirely different discussion!)

Good Luck!

Linda Sebree, HT
University of Wisconsin Hospital & Clinics
Department of Laboratory Medicine
IHC/ISH Laboratory
600 Highland Ave.
Madison, WI  53792-2472

Phone:  (608)265-6596
FAX:  (608)263-1568

> -----Original Message-----
> From: []
> Sent:	Wednesday, January 06, 1999 3:43 PM
> To:
> Subject:	ER/PR on cytological smears
> Does anyone know of a reason why ER/PR staining would appear as
> cytoplasmic and not nuclear when done on a smear? Any special protocols
> for doing this?
> Thanks
> Bev Miller

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