Hi,

I have tried 2 commercially available mGlur6 antybodies. But none of them worked. So, I was wondering if there is something wrong with my staining protocol.

samples: normal mouse retina.

fixation: 4% PFA perfusion or 4% PFA at 4 degrees for 1 hour.

Cryosections were washed by 0.1% triton X-100 and 0.05% tween-20 for 10 min each.Blocking reagent was 5% Donkey serum.Antibodies, including secondary antibodies, were diluted in a commercial antibody diluent. After incubating the first antibody overnight, I washed the samples with 0.05% tween-20 for 30 min at room temperature (RT) and then incubate them with Alexa Fluor 488 Donkey Anti-Goat IgG (H+L) Antibody for 1h at RT. Finally  samples were washed by 0.05% tween-20 for 30 min at RT.

If you have any suggestion, please tell me.

Thanks.