John,
I have seen some antibodies showing more background staining at higher
temperatures e.g. S-100. (Polyclonal). I found that e.g. ER and PR reacted
one degree weaker at high temperature than according to normal room
temperature. You could incubate at e.g. 4°C, but I advise you to look into
your antibody dilution. I think it is also important to let the reagents
come to room temperature for e.g. retrieval buffers, as you may loose some
of your heating capacity when using a standard protocol. For me also, those
are personal findings and not established studies.
Best regards,

Donald Van Hecke
Lab. Ana Path
St. Lucas - Brugge
Belgium

> -----Oorspronkelijk bericht-----
> Van:	Dawson, Glen [SMTP:GDawson@Milw.Dynacare.com]
> Verzonden:	woensdag 12 december 2001 16:35
> Aan:	Auld, John; 'HistoNet Server'
> Onderwerp:	RE: Her-2/neu
> 
> John,
> 
> Although I haven't carried out a scientific study by any means, I have
> noticed a trend towards darker staining when the IHC reagents and the wash
> buffer are at room temp. as opposed to fresh out of the refridgerator.  I
> use only room temp wash buffer on my routine IHC runs but always store my
> HercepTest wash buffer in the fridge.  If I forget to take this wash
> buffer
> out early to warm and perform staining using wash buffer at roughly 4
> degrees C, staining intensity is drastically reduced.  These results are
> not
> surprising nor are they inconsistent with the way one would expect any
> reactions to be affected due to temperature variances.  DISCLAIMER: THESE
> ARE NOT CONTROLLED STUDIES.  THESE ARE PERSONAL EXPERIENCES ONLY AND I AM
> NEITHER BASHING NOR ENDORSING ANY PRODUCTS OUT THERE.  
> 
> Glen Dawson
> 
> -----Original Message-----
> From: Auld, John [mailto:John.Auld@rfh.nthames.nhs.uk]
> Sent: Wednesday, December 12, 2001 7:25 AM
> To: 'HistoNet Server'
> Subject: RE: Her-2/neu
> 
> 
> 	Glen
> 
> 	I am interested in your comments about temperature of the reagents.
> We did a little experiment on how temperature affected immunostaining. My
> lab in not air 	conditioned and he temperature varies uite a bit,
> between 14
> and 33 C. I stained some slides at 14, 24, and 33 to assess what if any
> effect this had. I expected 	stronger staining more background at the
> higher temp, BUT  to my surprise  ER, at low and high temperatures,
> stained
> less cels and stained them weaker. These 	were not in line with a
> mislabelling and a few were repeated to check.
> 
> 	Does anyone have any suggestions or seen similar
> 
> 	John Auld FIBMS MSc
> 	Biomedical Scientist 3
> 	Immunocytochemistry
> 	Department of Histopathology
> 	Royal Free Hospital
> 	Pond Street
> 	London NW3 2QG
> 	Tel 020 7794 0500 ext. 6516
> 
> 	
> 
> 
> > -----Original Message-----
> > From:	HistoNet Server [SMTP:histonet@pathology.swmed.edu]
> > Sent:	Wednesday, December 12, 2001 7:47 AM
> > To:	HistoNet Server
> > Subject:	Daily Digest
> > 
> > 
> > 
> > Date: 11 Dec 2001 15:27:55 -0600
> > From: "Dawson, Glen" 
> > Subject: RE: Her-2/neu
> > 
> > Cathy,
> >  
> > I am currently doing FISH correlations on a number of cases done both
> here
> > and elsewhere in which the patient tissue was a 3+ on core biopsies but
> > were
> > negative on subsequent big blocks using the DAKO HercepTest kit.  I am
> > also
> > monitoring the results closely on all HercepTest cases that I perform.
> > Preliminary results are leading me to believe that the temperature of
> both
> > the Kit reagents as well as the HercepTest wash buffer are a much bigger
> > factor than I had believed.  There is a standing order here (Medical
> > College
> > of Wisconsin & Froedtert Hospital) to no longer do this test on breast
> > core
> > bx's until this problem is resolved.  I'll let you know what I find out.
> >  
> > Glen A. Dawson  BS, HT & IHC (ASCP)
> > Lead IHC Technologist
> > Milwaukee, WI
> > 
> > - -----Original Message-----
> > From: Cathy Thornton [mailto:cthornton@usiw.net]
> > Sent: Tuesday, December 11, 2001 9:17 AM
> > To: histonet
> > Subject: Her-2/neu
> > 
> > 
> > Is anyone using anything other than Herceptest for clinical Her-2/neu
> > stains.  If so what antibody and detection and could you discuss scoring
> > and
> > reporting the results??  Also has anyone done FISH correlations with IHC
> > results within your own laboratory?
> >  
> > Pat Zeitlow
> > Boyce and Bynum Pathology Laboratories
> > Columbia, Missouri
> > 
> > 
> >