Mouse anti map2ab

Cells were fixed in 4% paraformaldehyde in PBS at 4 °C for 10 min, permeabilized at room temperature for 15 min in 1.0% Triton in PBS and blocked in 5% donkey serum with 0.1% Triton at room temperature for 30 min. The following primary antibodies and dilutions were used: goat anti-Nanog (R&D, AF1997), 1:200; mouse anti-Tra1-60 (Millipore, MAB4360), 1:100; mouse anti-human Nestin (Millipore, ABD69), goat anti-Sox2 (Santa Cruz, sc-17320), 1:200; rabbit anti-βIII-tubulin (Covance, PRB-435P), 1:200; mouse anti-MAP2AB (Sigma, M1406), 1:200; mouse anti-S100b (Sigma-Aldrich, S2532), 1:1000. Secondary antibodies were Alexa donkey anti-rabbit 488 (Jackson Immuno 711-545-152) and 568 (Life Technologies A10042), Alexa donkey anti-mouse 488 (Jackson Immuno 715-545-151) and 568 anti-mouse (Life Technologies A10037), and Alexa donkey anti-goat 488 (Jackson Immuno 705-545-147) and 568 (Jackson Immuno 705-605-147); all were used at 1:300. To visualize nuclei, slides were stained with 0.5 μg ml⁻¹ DAPI (4′,6-diamidino-2-phenylindole) and then mounted with Vectashield.

Human iPSC-derived motor neuron cultures were plated on optical-bottom 96-well plates (Thermo, # 165305) and subsequently fixed in 4% paraformaldehyde for 15 minutes. Cells were blocked in 5% normal donkey serum with 0.1% Triton X-100 in PBS and incubated with primary antibodies for 1 h at room temperature or overnight at 4°C. Cells were then rinsed and incubated in species-specific AF488, AF594, or AF647-conjugated secondary antibodies followed by Hoechst 33258 (0.5 μg/mL; Sigma) to counterstain nuclei. Cells were imaged using Molecular Devices ImageExpress Micro high-content imaging system or using Leica microscopes (Fuller et al., 2015 (link)) (Figure 1B). Primary antibodies used were as follows: mouse anti-SMI32 (Covance, 1:1,000); mouse anti-TuJ1 (β3-tubulin) (Sigma; 1:1,000-1:2,000); rabbit anti-GFAP (Dako; 1:1000); mouse anti-Map2a/b (Sigma; 1:1000); rabbit anti-nestin (Millipore; 1:2000), Islet-1 Antibody (R&D AF1837; 1:250) and Nkx-6.1 (DSHB F55A10-s; 1:100).

Cells cultured on coverslips in 24-well plates (Costar) were fixed in 4% paraformaldehyde (PFA, Sigma) for 20 min at RT, washed twice in 0.15 M Sørensen’s buffer for 15 min and then permeabilized by washing three times in 0.1% Triton X-100 (Sigma) in 0.05 M TBS for 15 min at RT, followed by blocking in 5% goat serum (Millipore) in 0.05 M TBS for 30 min at RT. Afterwards, primary antibodies diluted in 5% goat serum were added to the cells in the following concentrations: rabbit anti-TH (Millipore #AB152) 1:600, mouse anti-MAP2a + b (Sigma #M1406) 1:2000, rabbit anti-LAMP1 (Abcam #108597) 1:1000, rabbit anti-TOM20 (Santa-Cruz #SC-11415) 1:1000. The cells were washed three times in 0.1% Triton X-100 in 0.05 M TBS for 15 min at RT and incubated with fluorophore-conjugated secondary antibodies: Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen #A11008), Alexa Fluor 488 goat anti-mouse IgG (Invitrogen #A11001), Alexa Fluor 555 goat anti-rabbit IgG (Abcam #150078) or Alexa Fluor 555 goat anti-mouse IgG (Molecular probes #21422) 1:500 diluted in 5% goat serum in 0.05 M TBS for 2 h at RT. The cells were washed twice in 0.05 M TBS for 15 min at RT and then counterstained with 10 µM DAPI (Sigma) for 15 min at RT to stain all nuclei. Finally, cells were mounted onto glass slides using ProLong® Diamond mounting medium (Molecular Probes).