Alexa 647 conjugated goat anti mouse secondary antibody

Tissues from transgenic marmosets were fixed in 4% paraformaldehyde (PFA) in PBS overnight at 4 °C. Tissues were embedded in OCT compound, and sliced into 40 μm sections. Alexa488 conjugated rabbit anti-GFP antibody (Invitrogen, 1:1,000) was used to enhance the GCaMP fluorescence and costained with an antibody against the neuronal marker NeuN (EMD Millipore, 1:500). Briefly, sections were incubated with the blocking buffer (5% normal donkey, and 0.2% Triton X-100 in 1X PBS) for 1 hr at room temperature and then incubated with Alexa488 conjugated rabbit anti-GFP antibody and mouse anti-NeuN antibody overnight at 4 °C. After incubation with the first antibody, sections were washed with 1X PBS three times for 20 min each, followed by incubation with Alexa 647-conjugated goat anti-mouse secondary antibody (Invitrogen) for 1 hr at room temperature and then washed with 1X PBS. Sections were transferred onto slides, mounted with 0.1% paraphenylinediamine in 90% glycerol/PBS, and imaged with confocal laser-scanning microscope LSM 5 Pascal (Carl Zeiss). AlexaFluor 488 and 647 were excited with 488 and 633 nm laser beams and observed through 510–530 and X650-nm emission filters, respectively.