Lsm 710 sim

Aliquots of 100 μl/well from 50 × 10⁵ cells/ml suspensions were cultured in 8 chamber polystyrene vessel tissue culture treated glass slides (Falcon, New York, NY) overnight or until confluence. Cells were then left untreated (control) or treated with tryptase (50 nM) for 48 h under culture conditions (37 °C in 5% CO₂). Then, supernatants were removed carefully and cells were fixed with 4% paraformaldehyde in PBS for 15 min. One hundred microliters of 50 μg/ml digitonin solution in PBS was added to each individual glass and incubated for 10 min at room temperature, followed by washing three times with TBS-T. The slides were kept in the dark and 50 μl of 1 μg/ml DAPI in TBS/1% BSA was added for 2 min, followed by three times washing with TBS-T. The slides were mounted with SlowFade® gold antifade mounting medium (Life Technologies). Samples were analyzed using a laser-scanning microscope equipped with ZEN 2009 software (LSM 710 SIM; Carl Zeiss, Berlin, Germany).

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.