The following antibodies were used for this study: rat anti-α-Tub (Serotec; 1:1,000), mouse anti-α-Tub (DM1A, Sigma; 1:2,500), rabbit anti-Asl (1:500), rabbit anti-Plp (1:1,000; gifts from J. Raff). Secondary antibodies were from Molecular Probes and the Jackson Immuno laboratory.
Ninety-six to one-hundred twenty hours after egg laying (AEL) larval brains were dissected in Schneider’s medium (Sigma) and fixed for 20 min in 4% paraformaldehyde in PEM (100 mM PIPES [pH 6.9], 1 mM EGTA and 1 mM MgSO4). After fixing, the brains were washed with PBSBT (1X PBS, 0.1% Triton-X- 100, and 1% BSA) and then blocked with 1X PBSBT for 1 h. Primary antibody dilution was prepared in 1X PBSBT, and brains were incubated for up to 2 days at 4°C. Brains were washed with 1X PBSBT 4 times for 20 minutes each and then incubated with secondary antibodies diluted in 1X PBSBT at 4°C overnight. The next day, brains were washed with 1X PBST (1x PBS, 0.1% Triton-X- 100) 4 times for 20 minutes each and kept in Vectashield H-1000 (Vector laboratories) mounting media at 4°C.
Ninety-six to one-hundred twenty hours after egg laying (AEL) larval brains were dissected in Schneider’s medium (Sigma) and fixed for 20 min in 4% paraformaldehyde in PEM (100 mM PIPES [pH 6.9], 1 mM EGTA and 1 mM MgSO4). After fixing, the brains were washed with PBSBT (1X PBS, 0.1% Triton-X- 100, and 1% BSA) and then blocked with 1X PBSBT for 1 h. Primary antibody dilution was prepared in 1X PBSBT, and brains were incubated for up to 2 days at 4°C. Brains were washed with 1X PBSBT 4 times for 20 minutes each and then incubated with secondary antibodies diluted in 1X PBSBT at 4°C overnight. The next day, brains were washed with 1X PBST (1x PBS, 0.1% Triton-X- 100) 4 times for 20 minutes each and kept in Vectashield H-1000 (Vector laboratories) mounting media at 4°C.