Re: HIER Methods Survey

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From:Phyllis Davie <pdavie@phenopath.com>
To:Luis Chiriboga <luis.chiriboga@med.nyu.edu>, Histonet <Histonet@pathology.swmed.edu>
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>Dear Histonetters:
>I am taking an informal survey to see what HIER methods/techniques
>everyone is using.  As with any survey,  the more responses the better.
>If anyone has additional questions they would like to add please e-mail
>me directly and i will reissue a questionnaire.
>
>1) Which heating method do you use for HIER? (i.e.. Microwave, pressure
>cooker, combination etc...)
     I have used all of the following:
          microwave
          microwave pressure cooker
          water bath
          steamer
     Having tried all of the above, I am of the opinion that ANY heating 
method can be made to be equivalent to any other, but that the times may 
need to be altered (sometimes by quite a lot) in order to do so.
>2) What is the average "real" heating time for antigen retrieval?
>(actual boiling time)
     This depends entirely on the method of heat delivery:
          microwave:               10-30 minutes
          microwave pressure cooker    8-20 minutes
          water bath               20-120(!) minutes
          steamer                  20-60 minutes 
>4) How long is your cooling period?
     We've standardized to 20 minutes for simplicity's sake, but not all 
antibodies require cooling.
>5) Do you perform manual or automated IHC?
     Both  (manual:  Vector Elite ABC)  (automated:  DAKO Autostainer 
with DAKO LSAB+ detection)
>5) Do you prepare your own retrieval solutions or purchase commercial?
     We prepare our own.
>6) Do you use more than one HIER solution? (please list)
     Oh yeah.       
          10mM Citrate, pH 6.0
          0.5M Tris, pH 10.0
          3M Urea
          0.01M EDTA, pH 8.0

          Not to mention that we fairly commonly do a combination 
HIER/enzyme digestion as follows:      
HIER in Citrate for 20 minutes (in the steamer), 
20 minute cooldown, wash in PBS, 
digest in 0.01% (w/v in PBS) Pronase (Calbiochem cat# 53702) for 10 
minutes at room temp., 
wash in PBS.......

>7) Which solution do you use the most?
         10mM Citrate, pH 6.0
>
>I will post the stats as soon as I compile all the responses.
>Thanks in advance
>
>
>
>--
>Luis Chiriboga Ph.D HT(ASCP)QIHC
>Department of Pathology 4W27
>Bellevue Hospital
>NYU School of Medicine
>First Avenue & 27th Street
>New York, NY 10016
>(212)562-4667
>
>
>
>



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