RE: antibody testing

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From:Cynthia Favara <cfavara@atlas.niaid.nih.gov> (by way of histonet)
To:histonet <histonet@magicnet.net>
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Heike,
	I have done some of this before. Most times the investigator would
like a quick and dirty experiment. Do it on a paraffin block and see if it
works.
	 I would first find out how the slides were fixed. Formalin,
alcohol, acetone??? or some other way. If they are alcohol fixed you might
want to try a formalin fixation on frozen first. If the procedure worka on
formalin fixed frozen sections I would be inclined to run paraffin section
with and without antigen retrieval using pH6.0 citrate buffer. Hopefully
one of those would be positive if not you can proceed to antigen retrieval
with other solutions at different pH.
	Find out as much as possible from the person you are doing the work
with and let them know  what is involved, helpful when they don't get
results in short order. Don't try to do too much at one time. I have
learned the hard way to KISS [keep it simple simple]
	There are some good articles by Shi and many others a lit search
will be helpful. I have some references if you would like more specifics
let me know.
	Hope this is helpful

Cynthia Favara
Rocky Mountain Laboratories
903 S 4th Street
Hamilton, MT 59840
ph: 406-363-9317
FAX: 406-363-9286
e-mail: cfavara@nih.gov

> ----------
> From: 	Heike Grabsch[SMTP:h.grabsch@uni-koeln.de]
> Sent: 	Saturday, January 09, 1999 11:39 AM
> To: 	histonet@pathology.swmed.edu
> Subject: 	antibody testing
>
> I hope I will get some input on the following problem:
>
> I got several monoclonal antibodies against different epitopes of the
> VEGF-receptor which were produced by another lab and tested on frozen
> human tissue sections. I have not seen the slides myself. Now, they want
> our lab to test those antibodies on paraffin embedded material. On frozen
> sections they used a concentration of 5 > g/ml with the Vectastain
> ABC-Kit.
>
> My question is: is there a reasonable way or strategy how to test the
> antibodies or is it just trial and error? As I have no idea which
> concentration or which potential pre-treatment could be succcessful I am
> not sure where I should start.
>
> Any suggestions are highly appreciated.
>
> thanks for your help,
>
> Heike Grabsch, Germany
>




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