immunostaining of ovaries

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immunostaining of ovaries

Postby Opal » Sun Apr 21, 2013 10:33 pm

I'm trying to do immunostaining on ovaries while my protein is initially expressed in the nurse cells and it's later transferred to the oocyte this protein has a strong localization to the posterior region at the late stage oocytes (from stage 8th onward). My antibody is very clean and specific, I do get a nice signal in the nurse cells but I do not get any clear signal in the oocyte. It seems that the antibody does not penetrate the oocyte and stays at the surface. I am doing another staining in parallel with another antibody and that one works fine even for the late stage egg chambers. Does any body have a special protocol for permeabilizing late stage oocytes. After fixation in 4% paraformaldehyde (in PBS)+heptane, I permeabilize the ovaries in 1% triton-X100 for 1hour at room temperature but apparently this is not enough! I would appreciate any suggestions.
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