Anti vacht

TeNT was purified from C. tetani Harvard strain cultures and was kept at –80°C (45 (link)). When injected in vivo, the toxin was dissolved in physiological solution plus 0.2% gelatin (G2500, MilliporeSigma). An affinity-purified antiserum specific for TeNT-cleaved VAMP was obtained as recently described (23 (link)); anti-VAChT (1:500, 139 105), anti-intact VAMP-2 (1:500, 104 211), and anti-VGAT (1:500, 131 308) antibodies were purchased from Synaptic System; anti-GlyT2 (1:500, AB1773) was purchased from Chemicon; and α-bungarotoxin Alexa Fluor 488 conjugated (1:200, B13422) and anti–guinea pig Alexa Fluor 488 conjugated (1:200, A11073) were purchased from Thermo Fisher Scientific. Anti-rabbit Alexa Fluor 555 conjugated (1:200, A21428) was purchased from Life Technologies.

Antibodies used for immunostaining were anti-Tubb3 (Cell Signaling D71G9; 1:100), anti-Map2 (Sigma M9942; 1:200), anti-VACht (Synaptic Systems 139 103; 1:200), anti-Isl1 (Abcam ab109517; 1:1000), and anti-Hb9 (DSHB 81.5C10; 1:100). Differentiated cells were fixed in 4% paraformaldehyde for 20 min and then permeabilized with 0.1% Triton-X for 15 min. After primary incubation for 1 hr, samples were labeled with a secondary antibody conjugated to AlexaFluor647. Samples were co-stained with DAPI before imaging on a Nikon Eclipse TE2000-E microscope.

Cultured motoneurons were fixed with 4% paraformaldehyde for 20 min (10 min on ice, 10 min at RT), permeabilized with 1% (v/v) Triton X-100 in PBS washing three times for 5 min each time. Cultures were incubated with the following primary antibodies: anti-bassoon (mouse monoclonal, 1:250, Enzo); anti-SMN (rabbit polyclonal, 1:250, Santa Cruz); anti-vGLUT2 (rabbit policlonal, 1:250, Synaptic Systems); anti-PSD-95 (mouse monoclonal, 1:250, Millipore); anti v-AChT (rabbit polyclonal, 1:250; Synaptic Systems) diluted in 5% (w/v) BSA, 1% Triton X-100 in PBS for 1 h. After washing three times for 5 min with 1% Triton X-100 in PBS, cells were incubated for 1 h with the secondary antibody (Alexa 594 or Alexa 647-conjugated donkey anti-rabbit or anti-mouse, Invitrogen) in PBS 1X containing 0.05% Triton X-100 (1:500) and washed with PBS three times for 5 min. Finally, cells were mounted with slowfade medium (Invitrogen) on microscope slides.