Antibody incorporation problem

[Klaudia]

Hi,

I have a new antibody, and I'm trying to do immunofluorescence in drosophila larval brains, but my antibody don't penetrate the brain, it stays only in brain surface.
Someone can help me to find a way to do the antibody penetrate the brain??

Thank you.

[haisond]

Hello,

Where is your target antigen? is it localized on cell membrane, nucleus, or cytoplasm? if not localized on membrane, you should permeabilize with an ionic detergent such as Triton-x100 or Tween 20. Permeabilize your specimen with a 0.1% to 0.3% Triton-x 100 solution in your buffer of choice. This should punch holes in cell membrane to allow for the penetration of the primary abs.