IHC on cytology specimen

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From:"Sebree Linda A." <la.sebree@hosp.wisc.edu>
To:'Histonet' <histonet@pathology.swmed.edu>

> Hi Amy,
> First of all, to destain a PAP smear, remove the coverslip and hydrate to
> 70% alcohol.  Immerse in 1% HCl in 70% alcohol for several minutes.  You
> will see color running off the slide.  All of the dye may not come off but
> it eventually will during the immunostaining procedure.  Rinse well in
> several changes of water before going into APK.
> We stain our frozen tissues and cytology specimens on Ventana ES and genII
> instruments using a wetload, frozen protocol with a 28" titration, AB
> block and counterstain.  If the specimen is particularly bloody, you might
> want to use a paraffin protocol so it receives inhibitor.  We manually
> apply our chromogranin (2 dispenses of a 1:300 dilution of Dako's
> polyclonal antibody).  In a majority of frozen/cytology cases, we've found
> that we have to use a dilution twice as concentrated as for paraffin.
> Keep in mind that if you don't have a cytology or frozen tissue positive
> control, using a paraffin positive control and its staining protocol will
> only tell you that the antibody is working on paraffin.  If you get a
> negative result on your FNA smear be sure and let the pathologist know
> what type of specimen you ran as a positive control.  Not having a slide
> for a negative control can make interpretation difficult also, since after
> applying inhibitor and AB block you may still get endogenous staining that
> is difficult to discern from the "real" thing.
> Good Luck and if I can help in any other way, don't hesitate to call or
> e-mail.
> Linda A. Sebree, HT
> University of Wisconsin Hospital & Clinics
> Immunohistochemistry/In Situ Hybridization Laboratory
> D4/218-2472
> 600 Highland Avenue
> Madison, WI  53792-2472
> (608)265-6596
> FAX: (608)263-1568
> -----Original Message-----
> From:	Amy Self [SMTP:AmyS@gmhsc.com]
> Sent:	Wednesday, August 16, 2000 2:24 PM
> To:	'Histonet@Pathology.swmed.edu'
> Subject:	IHC on cytology specimen
> 	I have to do a chromogranin on a FNA parotid mass slide that has
> already been PAP stained.  What is the best way to destain this slide?
> Also,
> I am just learning immuno's and thank God my lab bought a Nexes from
> ventana. Does anyone have any suggestions/procedure about this stain that
> will help me out?  I have only done immuno's on paraffin sections and this
> is the ONLY slide that we have on this case.  If anyone can help please
> e-mail privately or call me at 843-527-7179.
> 					Thanks,
> 				                Amy

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