B 1035

Thick 100 μm cryostat sections were rehydrated in PBS. Sections and whole mount pancreata were blocked overnight in PBS, 2% donkey serum and 0.5% Triton-100X. The samples were incubated in primary antibodies (Chromogranin A, 1:180 from Abcam, ab15160; insulin, 1:100, from Abcam, ab7842; glucagon 1:200, from Abcam, ab10988); and Dolichos biflorus agglutinin (DBA) biotinylated (1:270, from Vectorlabs, B-1035) for 3 days at 4 °C. Secondary antibodies were applied (from Thermo Fisher Scientific) and AF647-Streptavidin (1:800, from Thermo Fisher Scientific, S21374), BV510-Streptavidin (1:600, from Biolegend, 405233) for 2 days at 4°C. All tissues were cleared with RapiClear 1.52 (from SunJin Lab, RC152001).
Thin sections were incubated in 2% Serum, 0.1% Triton-100X in PBS for 30 min, subsequently incubated in primary antibody in 0.01% Triton-100X in PBS overnight (insulin 1:100, from Abcam, ab7842, glucagon 1:200 from Abcam, ab10988, cleaved caspase 3, 1:400, from Cell Signalling, 9664S), washed 3 times for 10 min the next day, and incubated with secondary antibody in 0.01% Triton-100X for 3 h, washed and mounted.

HFFs confluent on coverglass were infected with 1 × 10⁴–5 × 10⁴ tachyzoites. After a four-hour invasion period at 37°C and 5% CO₂, the media was exchanged for DMEM supplemented with 10% FBS for spontaneous differentiation assays or RPMI with 1% FBS for alkaline stress differentiation assays. Spontaneous and alkaline stress differentiation assay samples were incubated at 37°C/5%CO₂ or 37°C/0% CO₂ for 48 h, respectively, before fixation in 4% formaldehyde in PBS for 10 minutes. The assays were incubated for 10 minutes at room temperature in blocking solution (1% bovine serum albumin in PBS). The wells were then incubated in a primary antibody blocking solution containing rabbit anti-GAP45 or guinea pig anti-CDPK1 or biotinylated dolichos (Vector labs B-1035) for 60 minutes at room temperature. Secondary staining was performed with streptavidin-APC and goat anti-rabbit Alexa fluor 488 or goat anti-guinea pig Alexa fluor 488 in blocking solution for 60 minutes at room temperature. Three PBS washes were performed between antibody incubations. Images were acquired with a widefield Nikon Ti epifluorescence scope with 20x objective, with 3 images per coverslip. The percentage of DBL+ vacuoles was determined by manual quantification of vacuoles with DBL staining as a proportion of vacuoles with GAP45 staining. Images included in figure panels were false-colored in FIJI.