Mouse anti hb9

Chick embryo electroporation was performed as described^{20 (link)}. Fertilized White Leghorn chicken eggs (Charles River Laboratories) were incubated at 38°C until embryos reached Hamburger and Hamilton (HH) stage 10. Eggs were windowed, and plasmid DNA resuspended at 2–5 μg/μl in PBS was injected into the neural tube of the embryo. Embryos were electroporated between 4 mm gap electrodes at 25 V, 5 × 25 ms pulses with an ECM-820 electroporator (BTX). After electroporation, embryos were incubated until they reached stage 18–20 HH, fixed, incubated in 30% sucrose/PBS overnight, mounted and frozen in OCT compound, and cryosectioned at 12–18 μm. Sections were washed 3x with PBS/0.01% TritonX-100 and blocked for 30 minutes with PBS/0.01% Triton X-100 in 5% donkey serum. Sections were immunostained with mouse anti-Pax7 (1:25), mouse anti-Hb9 (1:100) (both from the Developmental Studies Hybridoma Bank, Iowa City, IA), and rabbit anti-GFP (1:1000) (Invitrogen) for 2 hours at room temperature or overnight at 4°C. After washing with PBS/0.01% TritonX-100, the appropriate secondary antibodies added and were incubated for 1 hour. Images were obtained using routine fluorescence microscopy techniques on a Microphot-SA epifluorescence microscope (Nikon) or an Axio Observer Z1 (Zeiss, Jena, Germany) fluorescence microscope.

iMNs were fixed in 4% paraformaldehyde (PFA) for 1h at 4 ºC, permeabilized with 0.5% PBS-T overnight at 4 ºC, blocked with 10% FBS in 0.1% PBS-T at room temperature for 2 h, and incubated with primary antibodies at 4 ºC overnight. Cells were then washed with 0.1% PBS-T and incubated with Alexa Fluor® secondary antibodies (Life Technologies) in blocking buffer for 2 h at room temperature. To visualize nuclei, cells were stained with DAPI (Life Technologies) then mounted on slides with Vectashield® (Vector Labs). Images were acquired on an LSM 780 confocal microcope (Zeiss). The following primary antibodies were used: mouse anti-HB9 (Developmental Studies Hybridoma Bank); mouse anti-TUJ1 (EMD Millipore); rabbit anti-VACHT (Sigma); rabbit anti-C9ORF72 (Sigma-Aldrich); mouse anti-EEA1 (BD Biosciences); mouse anti-RAB5 (BD Biosciences); mouse anti-RAB7 (GeneTex); mouse anti-LAMP1 (Abcam); mouse anti-LAMP3 (DSHB, cat. no. H5C6); rabbit anti-LAMP3 (Proteintech, cat. no. 12632); mouse anti-LAMP2 (DSHB, cat. no. H4B4); mouse anti-M6PR (Abcam, cat. no. Ab2733); rabbit anti-GluR1 (EMD Millipore, cat. no. pc246); mouse anti-GluR1 (Santa Cruz); rabbit anti-NR1 (EMD Millipore); mouse anti-NR1 (EMD Millipore, cat. no. MAB363); chicken anti-GFP (GeneTex).