Immunocytochemical analysis of UNC93A in mouse cortical neurons

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.