Chicken anti β gal

Standard Hematoxilin & Eosin, Cresyl Violet and Luxol Fast Blue histochemical stainings were performed on paraffin sections and digitized using Mirax (Carl Zeiss) or Aperio (Leica) slide scanners as described previously [79 (link)]. Immunofluorescent detection was performed on free-floating sections as described [80 (link)] using the following antibodies: rabbit anti-ASPA serum (in house), mouse anti-NeuN (Merck), rat anti-MBP (Abcam), mouse anti-GFAP (Cell Signaling), chicken anti-β-Gal (Abcam), mouse anti-Flag (Cell Signaling) and rabbit anti-Neurofilament 200 (Sigma) followed by the appropriate Alexa-488/594 conjugated secondary antibodies (Thermo Fisher). Immunoperoxidase detection was performed on free-floating sections as described in [15 (link)] using mouse anti-APC (Merck) and rabbit anti-NeuN (Cell Signaling), the appropriate biotinolated secondary antibody (Dianova) and a Vectastain Elite ABC kit (Vector Labs). Immunoblotting was performed as described previously [78 (link)] with the following antibodies: rabbit anti-ASPA serum, mouse anti-GAPDH (Sigma), rat anti-MBP (Abcam), rat anti-PLP aa3 and rat anti-NG2 (gift of J. Trotter) followed by the appropriate HRP-conjugated secondary antibodies (Dianova). qRT-PCR for Aspa, Nat8l, NAAG synthetase-I and II (Rimklb and Rimkla) and hypoxanthine phosphoribosyltransferase (Hprt) was performed as described [26 (link)].

Immunostaining and in situ hybridization were performed as previously described [47 (link)]. The Fezf2 cRNA probe corresponds to nucleotides 644 to 1,374 of mouse Fezf2 (GenBank: NC_000080). LacZ staining was preformed according to standard protocols. Primary antibodies used: Chicken anti β-gal (Abcam, 1:500), Rabbit anti BLBP (Millipore, 1:500), Rat anti CTIP2 (Abcam, 1:1,000), Guinea Pig anti GFAP (Advaned Immuno Chemical, 1:100), Rabbit anti PAX6 (Covance, 1:100), PHH3 (Cell Signaling, 1:100), Rabbit anti SATB2 (Abcam, 1:1,000), Goat anti SOX2 (Santa Cruz Biotech, 1:500), Rabbit anti SOX5 (Abcam, 1:500), Rabbit anti TBR1 (Abcam, 1:1,000), Mouse anti Tuj1 (Covance, 1:1,000). Primary antibodies were detected using AlexaFluor-conjugated secondary antibodies (Invitrogen, 1:1,000). DNA was visualized with DAPI (1:50,000).

For immunohistochemistry, wild-type or vps35 lacZ KI [105] (link) mouse eyes were fixed in 4% paraformaldehyde in PBS for 45 min, washed in PBS, then stored overnight in 30% sucrose in PBS. The cornea and lens were removed, and eyecups were embedded in OCT (Tissue-Tek) and flash-frozen. Cryosectioned retina were fixed in 2% paraformaldehyde in 1× PBS for 10 min and stained with the indicated antibodies: chicken anti–β-GAL (abcam, 1∶1,000) and rabbit α-melanopsin (Advanced Targeting Systems, 1∶5,000). Alexa 488- or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch, 1∶600) were used for immunostaining. Images were captured using the Zeiss LSM 710 confocal microscope and analyzed using the Image J software.

Larval brains were fixed and stained as previously described [32 (link)]. The following primary antibodies were used: rabbit anti-Ase (1:1000 from Y.N. Jan), chicken anti-β-gal (1:100 abcam), mouse anti-Dl (1:100, C594.9B Developmental Studies Hybridoma Bank, DSHB), guinea pig anti-Dpn (1:5000, from J. Skeath), chicken and rabbit anti-GFP (1:2000 abcam), rat anti-L’sc (1:5000) and anti-Notch (1:50, C17.9C6 DSHB). Alexa Fluor conjugated secondary antibodies were diluted 1:200 (Molecular Probes, Invitrogen). Primary and secondary antibodies were incubated at 4 °C overnight.