Alexa 405 donkey anti guinea pig igg

For immunohistochemistry, fixed coverslips were incubated in blocking solution (3% normal donkey serum, 0.3% Triton X-100 in TBS) at room temperature for 1 hour. Primary antibodies, mouse anti-nestin (1:500, Abcam), guinea pig anti-synapsin1/2 (1:500; Synaptic Systems), rabbit anti-RFP (1:500; Rockland), or chicken anti-neurofilament (M+H; 1:1000; Aves Labs) were diluted in antibody diluent solution (1% normal donkey serum in 0.3% Triton X-100 in TBS) and incubated overnight at 4°C. Samples were then rinsed three times in TBS and incubated in secondary antibody solution (Alexa 488 donkey anti-chicken IgG; Alexa 594 donkey anti-rabbit IgG, Alexa 647 donkey anti-mouse IgG, and Alexa 405 donkey anti-guinea pig IgG all used at 1:500 (Jackson Immuno Research)) in antibody diluent solution at 4°C overnight. Coverslips were rinsed three times in TBS and mounted with ProLong Gold Mounting medium (Life Technologies). Images were collected using a 63x oil-immersion objective on a Zeiss LSM800 confocal microscope and processed with airyscan. Coverslips were used to quantify colocalization as described below.