In situ hybridizationSections were dried for 1 hour at 60°C, pretreated and hybridized with an hs-ITPR2 probe (Homo sapiens inositol 145-trisphosphate receptor type 2 (ITPR2) mRNA) for 2 hours at 40°C. Probes were custom-designed and labeled for use with RNAscope 2.5HD (Advanced Cell Diagnostics, Newark, CA, USA). Some sections were stained with Hs-PPIB or dapB probes as positive and negative controls, respectively. Amplification steps were performed prior to the detection of signals with 3,3′-diaminobenzidine. Sections were counterstained and mounted with Permount (Fisher scientific, Waltham MA, USA).

ImmunofluorescencePancreatic slides were deparaffinized with Slidebrite (BioCare Medical, Concord CA) and dehydrated using graded ethanol concentrations. Slides were boiled in antigen retrieval citrate buffer pH6, blocked for 1hr with 2% normal goat serum, 2% bovine serum albumin, 0.5% Tween 20 in phosphate buffer saline followed by incubation with primary antibodies overnight at 4°C using different combinations. The staining series included antibodies to IP3R2 (Abcam, Cambridge, MA, USA 1:50), Translocase of outer mitochondrial membrane 20 (TOM20) (Santa Cruz biotechnologies, Dallas Texas, USA 1:50), VDAC-1 (Abcam, 1:100), mitofusin-2 (Abcam, 1:100), insulin (Dako, Carpinteria, CA, USA 1:150), glucagon (Abcam, 1:200). Secondary antibodies coupled to a fluorochrome (AF488, AF555, AF647, Life Technologies, Grand Island, NY, USA) were added for 30 min at RT according to the species of primary antibodies. Nuclei were stained with Hoechst 33342 (Sigma-Aldrich) and preparations were mounted in Prolong Gold anti-fade reagent (Life Technologies). Slides were analyzed with a slide scanner AxioScan.Z1 (Carl Zeiss SAS, Marly le Roi, France) at x40 magnification.

In situ proximity ligation assayDuolink II in situ proximity ligation assay (PLA) (Olink Bioscience, Uppsala Sweden) enables detection, visualization, and quantification of protein interactions (<40 nm) as an individual dot by microscopy. Primary antibodies to assess ER-mitochondria interactions were against IP3R2 (1:100) and VDAC-1 (1:200) as previously described [7]. Digitized slides were analyzed at x20 magnification. When necessary, beta cells and alpha cells were identified using anti-insulin and anti-glucagon antibody staining on the same slide. Dots were quantified in each islet using the Zen program and Fiji-ImageJ software and expressed as percentage of dots per nucleus. Experiments were performed at least twice using 2 to 5 non-consecutive slides for each donor. For Min6-B1 cultures, ER-mitochondria-interactions were assessed using a fluorescent PLA assay, employing antibodies directed against VDAC-1 (Abcam, 1:100) and IP3R1 (Santa Cruz laboratories, Dallas TX, USA 1:500) as described previously [10]. Experiments with Min6-B1 cells were performed at least three times, with a minimum of five fields taken per condition.