Duolink in situ orange kit

Proximity Ligation Assays (PLAs) to detect IP3R1-VDAC1 or VAPB-PTPIP51 interactions were performed as described previously using Duolink In Situ Orange Kits (Sigma) according to manufacturer’s instructions [17 (link), 18 (link), 35 (link)]. After amplification steps, samples were immunostained with β-tubulin III (SHSY5Y cells) or MAP2 (neurons) to confirm neural identity.
To detect serine-9 phosphorylated GSK3β, cells were fixed in 4% paraformaldehyde, washed three times in Tris-HCl buffered saline (TBS), permeabilised with 0.1% Triton X-100 in TBS for 15 min and blocked in 5% bovine serum albumin (BSA) in TBS for 1 h. Cells were then labelled with primary antibody diluted in 1% BSA in TBS overnight at 4 °C, washed three times with 0.01% Triton X-100/TBS, incubated with secondary antibodies diluted in TBS, washed three times with TBS and mounted in aqueous mounting medium with DAPI (Abcam). Z-plane images with 0.3 μm intervals were captured using a Nikon Eclipse Ti-E Inverted microscope with CFI Apo Lambda S 60x/1.40 objective and an Andor iXon EMCCD camera equipped with Visitech iSIM Super Resolution System. Mean cytoplasmic fluorescent intensities were quantified using ImageJ.

Neurons grown on coverslips were fixed for 15 min at 20 °C with 4% (w/v) paraformaldehyde in PBS and then permeabilized with PBS containing 0.5% Triton X-100 for 15 min. Samples were then preincubated with blocking buffer (PBS containing 10% goat or 2% donkey serum and 0.5% Triton X-100) for 1 h and incubated with primary antibodies diluted in blocking buffer for 16 h at 4 °C. Following washing in PBS containing 0.5% Triton X-100, the samples were incubated with goat/donkey anti-rabbit, mouse, rat or chicken Igs coupled to AlexaFluor − 488, − 594 or 647 in PBS for 1 h, washed in PBS and then mounted in Vectashield mounting medium (Vector Laboratories). Proximity ligation assays (PLAs) to identify the VAPB-PTPIP51 interaction were performed essentially as described previously using Duolink reagents (Sigma-Aldrich) [13 (link)]. Briefly, neurons were fixed in 4% paraformaldehyde in PBS and probed with rat anti-PTPIP51 and rabbit anti-VAPB antibodies, and signals developed using a Duolink In Situ Orange kit (Sigma-Aldrich). Following PLAs, neurons were immunolabeled for synaptophysin and PSD95.