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Thank you for your kind reply.
please find attchaed protocol that you requested.
I have tried different concentrations and incubation times but obsrved the same
I am looking forward to hearing from you.
Asked on Mar 15 2012
Thank you for your reply with this information.
Regarding your protocol - one thing that really stood out is the use of Triton as a permeabilizing agent. This detergent will also partially dissolve the nuclear membrane and will disrupt proteins when used at higher concentrations for longer periods of time. We typically recommend using it at 0.1-0.2% for 10 min only. Since both of these proteins are cytomplasmic, permeabilization with tween 20 is sufficient (I recommend 0.2 % for 10 - 15 minutes). This should decrease the amount of background and non-specific stain you are observing.
Additionally, I would recommend optimizing the conditions with each antibody individually rather than in sequential staining.
I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions or concerns.
Answered on Mar 15 2012