Ab12370

Wildtype male and female mice were mated and at e14-15 the females were euthanized with cervical dislocation, the embryos removed and cortex dissected out and primary cultures were set up as previously described in Perland et al. (2016) (link). Immunocytochemistry was performed as described in Perland et al. (2016) (link) with anti-UNC93A diluted 1:100 in supermix blocking solution. Co-staining with neuronal marker Pan diluted 1:200 (MAB2300, Millipore), KDEL markers (ab12223, Abcam) diluted 1:200, Syntaxin 6 (Ab12370, Abcam) diluted 1:100, Synaptotagmin (ab13259, Abcam) diluted 1:200 and SNAP25 (ab25737, Abcam) diluted in 1:100 in supermix blocking solution. Images were acquired at the SciLifeLab BioVis Facility (Uppsala University) using confocal LSM710 SIM (Zeiss) and the Zen black software (Zeiss) or Olympus microscope BX55 with an Olympus DP73 camera and the cellSens Dimension v1.14 (Olympus). Images were then handled using ImageJ, Fiji edition (Schindelin et al., 2012 (link)).
In addition, immunocytochemistry for the N-terminal anti-UNC93A antibody (ab173552, Abcam), diluted 1:80 in 5% milk block (Bio-Rad), was performed as described above.

Brains from mice at 61–65 days were dissected on the sagittal plane and flash frozen. Following weighing, samples were ribolysed in Dulbecco’s phosphate-buffered saline (DPBS) with ceramic homogenisation beads (Bertin Technologies) at max speed for 105 s to produce 10 and 20% homogenates which were stored at − 80 °C until use.
Homogenates were diluted in DPBS with 4× SDS sample buffer (250 mM Tris base, 40% glycerol, 8% SDS, 0.04% bromophenol blue) and boiled at 95 °C for 5 min. 35 μg of total protein was loaded onto a 4–12% (w/v) Bis-Tris polyacrylamide gel (Invitrogen) and electrophoresed before being electroblotted to a nitrocellulose membrane. Membranes were blocked in Odyssey Blocking Buffer for 1 h at room temperature (RT), before incubation with anti-syntaxin-6 (1:500 clone C34B2 (Cell Signalling; 2869S) or clone 3D10 (Abcam; Ab12370)) overnight at 4 °C. Membranes were washed with 0.05% Tween-20 in phosphate buffered saline (PBST) for 3 × 5 min with agitation and incubated with anti-β actin (1:1000 rabbit polyclonal (Sigma Aldrich; A2066) or 1:5000 mouse monoclonal (Sigma Aldrich; A5441)). After washing, membranes were probed with fluorophore-conjugated secondary antibodies (IRDye 800CW Donkey anti-rabbit IgG and IRDye 680RD goat anti-mouse IgG both at 1:4000) for 1 h at RT. Membranes were washed again and imaged with Odyssey Gel Documentation System (LI-COR; Model 9120).