Rabbit anti gfp primary antibody

Cortical neurons plated on slides were rinsed three times in ECS, fixed in 4% paraformaldehyde for 5 min, and blocked with 5% BSA in ECS for 10 min. For surface staining, the neurons were incubated in rabbit anti-GFP primary antibody (Abcam, 1:500) for 15 min, rinsed three times in ECS, and incubated with Alexa-546-conjugated secondary antibody (Abcam, 1:1 000) for another 15 min. For intracellular staining, next the neurons were blocked and permeabilized with 0.1% Triton X-100 in PBS containing 5% BSA, then incubated in primary and secondary antibodies for 1 hr each with three times of rinse. Then the neurons were then examined under a 60×, 1.4 numerical aperture oil-immersion objective on an Olympus confocal microscope equipped with FV1000 software. Surface and intracellular staining were analyzed using Metamorph 5.0 software, and the ratio of surface/intracellular signals after stimulation was normalized to the ratio in transfected neurons before stimulation.