Alexa568 conjugates

Anti-CD200 (R & D Systems, Minneapolis, MN, AF2724), anti-Shh (Santa Cruz Biotechnology, Santa Cruz, CA, sc-9024), anti-Bmi-1 (clone F6, Millipore Corp., Billerica, MA, 05-637), anti-β actin (Sigma-Aldrich, St. Louis, MO, A2228), and anti-GAPDH (Ambion, Austin, TX, AM4300) were used for immunoblotting, immunocytochmistry, and immunohistochemistry. As secondary antibodies, HRP-conjugated anti-rabbit (GE Healthcare, Piscataway, NJ, NA934), anti-mouse (R & D Systems, HAF007), and anti-goat (Santa Cruz Biotechnology, sc-2020) were used for western blot analysis. Secondary antibodies for immunofluorescent localization were as follows: Alexa conjugates [anti-mouse (A11001); anti-goat (A11055)], and Alexa568 conjugates [anti-rabbit (A11011); anti-mouse (A11019)] were from Invitrogen Co (Carlsbad, CA).

Flat-mount preparations were cryoprotected (1 hr 10% sucrose in PBS at room temperature [RT], 1 hr 20% sucrose in PBS at RT, and overnight 30% sucrose in PBS at 4 °C), frozen and thawed three times, and blocked with 10% normal donkey serum in PBS for 2 hr before incubation with primary antibodies for five days at 4 °C. Flat mounts were washed in PBS (3 × 1 hr) at RT, incubated with secondary antibodies for one day at 4 °C, and washed in PBS (3 × 1 hr) at RT. Brain slices were blocked with 10% normal donkey serum in PBS for 2 hr before incubation with primary antibodies for 3 hr at RT, washed in PBS (3 × 20 min) at RT, incubated with secondary antibodies for 2 hr at RT, and washed in PBS (3 × 20 min) at RT. The following primary antibodies were used in this study: mouse anti-RFP (1:1,000, Abcam), guinea pig anti-RBPMS (1:1000, PhosphoSolutions), rabbit anti-Serotonin (1:200, S5545, Sigma–Aldrich). Secondary antibodies were Alexa 488 and Alexa 568 conjugates (1:1,000, Invitrogen).