Immunos on mouse tissues reply

From:Gayle Callis

You wrote. 
I have some mouse tissues (placentas, embryos mainly) I am trying to do an
immuno with an antibody raised in goat. I am using Sigma's Extravidin kit
and noticed their anti-goat biotin was raised in mouse. Is this going to
cause me problems? I have successfully used mouse primary on mouse tissues,
but never tried a secondary. 
A second question; do people recommend doing antigen retrieval? I tried
doing pepsin digestion and I got no signal in my immunos. 
Your input would be much appreciated. 


Hugo Caldas
Columbus Children's Hospital
Columbus, OH

Yes, their secondary will create problems as it will recognize other mouse
tissue antigen receptors, and you will get background. Buy an antigoat
antibody, Donkey antigoat should work, and make sure it is adsorbed to
mouse tissues, rabbit antigoat should work also adsorbed to mouse tissues.
Jackson Immunoresearch has an excellent selection of secondary antibodies,
there are other sources also. You should be able to substitute a mouse
antigoat antibody with a donkey antigoat or rabbit antigoat, just pay
attention to your normal serum blocking.  If you use VECTORS Elite ABC kit,
universal, you use any secondary antibody you want, may be a better kit as
it is less restrictive.  Check out their website for details, they also
have the protocol there.   

Also, if your primary antibody is BIOTINYLATED, you can also avoid your
secondary binding to mouse tissues.  DAKO ARK kit will do this for you, and
it comes complete with blocking reagents, easy to use! 

It may take a battery of retrievals or digestions, including your pepsin,
trypsin, etc to optimize your antibody staining.  

How you fix and process your tissue can determine success, and it may be
that your antigens are not retrievable because of the way you have fixed
the tissues initially, change fixationg or you may have to do frozen
sections.  You did not say what you were staining for??? 


Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


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