Re: mice frozen tissue IHC protocol.

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From:"Tim Morken" <timcdc@hotmail.com>
To:HistoNet@Pathology.swmed.edu
Reply-To:
Date:Mon, 05 Apr 1999 08:43:36 EDT
Content-Type:text/plain

Masayuki Miyagishima, MD,

<1) If you have choice,  which primary antibody do you chose, rat, 
rabbit
or goat, etc?>
1) An rat primary may cross-react with mouse tissue. I'd use anything 
besides rat or mouse primary.

<2) Again, which secondary antibody do you chose?  For example, if you
choose rabbit primary antibody, do you choose goat anti-rabbit or rat
anti-rabbit?
2) The same as with the primary. Stay away from rat or mouse.


<3)If you go for fluorescense, which fluorescent conjugated secondary is
your choice, Alexa, Fit-C, Cy3, etc?>
3) the fluoroscine tag you use depends on the filters you have for your 
microscope. Otherwise, use what you like and what is available.

4) If you go for peroxidase, which secondary is your choice (product
name?)?
4) the enzyme label is up to you. A peroxidase label will allow for more 
colors. The Alkaline phosphatase label is more sensitive, but you are 
more limited in colors. We use alk phos with fast red chomogen (all from 
DAKO)

5) For blocking solution, what is your strategy?
5) we use 20% sheep serum for 15 minutes. You may use something 
different or try a non-protien blocking agent. Use 3% hydrogen peroxide 
for blocking endogenouse peroxidase.

Am I asking too much??

Not at all!


Tim Morken, B.A., EMT(MSA), HTL(ASCP) 
Infectious Disease Pathology
Centers for Disease Control
MS-G32
1600 Clifton Rd.
Atlanta, GA 30333
USA

email: tim9@cdc.gov
       timcdc@hotmail.com

FAX:  (404)639-3043

-- 
Masayuki Miyagishima, MD
University of Pittsburgh, 
Department of Surgery
412-647-2345, and ask the hospital operator to page 3228, please.



Tim Morken, B.A., EMT(MSA), HTL(ASCP) 
Infectious Disease Pathology
Centers for Disease Control
MS-G32
1600 Clifton Rd.
Atlanta, GA 30333
USA

email: tim9@cdc.gov
       timcdc@hotmail.com

FAX:  (404)639-3043


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