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Position: Discussion forums > Bioscience biotechniques discussion forums > Immunohistochemistry In Situ Hybridization forum
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monoclonal GFAP immunostaining
Posted by: lee2245 (IP Hidden, Junior member, 12)
Date: June 9, 2005 09:58PM
Anyone has experiance of blood vessel staining with mouse monoclonal primary antibody with mouse brain tissue? I guess it is nonspecific binding of secondary antibody??
I tried to use sheep anti-mouse IgG to block the nonspecific binding of goat anti-mouse IgG, but it didn't help me at all. Any suggestions will be helpful. Have a great day!
Re: monoclonal GFAP immunostaining
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: June 10, 2005 01:59PM
You should use "normal goat serum" for blocking, test 2%, 10%, 20%...upto 50% to see if you can reduce background. The background issue may be from primary antibody too, test different dilution of the primary may help.
Re: monoclonal GFAP immunostaining
Posted by: Nick2321 (IP Hidden, Unregistered user, )
Date: June 10, 2005 04:33PM
Hi,
Did you try Vector Mouse on Mouse (MOM?) Kit before? To exclude the possibility of secondary non-specific binding you can include a control without primary antibody incubation. Good luck. Nick.
Re: monoclonal GFAP immunostaining
Posted by: lee2245 (IP Hidden, Junior member, 12)
Date: June 27, 2005 11:41AM
Thanks, the MOM kit works.
Re: monoclonal GFAP immunostaining
Posted by: lee2245 (IP Hidden, Junior member, 12)
Date: June 27, 2005 11:43AM
By the way, when you do triple labelling, do you incubate primary or secondary antibodies sequencially or you incubate three antibodies all together at the same time?
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