A1 point scanning confocal microscope

FFPE pancreas sections of 4 µm thickness were dewaxed and rehydrated according to standard protocols. The sections were immersed in a sodium citrate buffer (10 mM sodium citrate, pH 6.0) and subjected to heat-induced epitope retrieval. After rehydration in PBS, the sections were treated with a serum-free protein block (Dako, North Sydney, NSW, Australia) for 20 min, followed by primary antibody incubations at 4 °C overnight using guinea pig anti-insulin (1:200, Abcam, Melbourne, VIC, Australia), mouse anti-glucagon (1:200, Abcam, Melbourne, VIC, Australia), and rabbit anti-somatostatin (1:250, Abcam, Melbourne, VIC, Australia), all diluted in antibody diluent (Dako, North Sydney, NSW, Australia). Visualisation was performed using Alexa Fluor dyes (Life Technologies, Scoresby, VIC, Australia) and was imaged using a confocal Nikon A1+ point scanning confocal microscope.

Agroinfiltrated N. benthamiana leaf tissue was sampled at 2–3 DPI, mounted on a microscope slide, and viewed on a Nikon A1+ point scanning confocal microscope (Nikon). To assess apoplastic localization, samples were incubated in 30% glycerol for at least 30 min to induce plasmolysis prior to mounting. GFP fluorescence and chloroplast autofluorescence were excited at 489 nm and emission was captured at 525 nm and 700 nm, respectively. Objective magnification of 10× or 20× was used.

Wild-type and Δdhgd-1 C. elegans strains were crossed to a strain with fluorescently labeled mitochondria and nuclei Pmyo-3::GFPmito; Pmyo-3::lacZ::GFP(nls) in body wall muscles. Animals maintained on E. coli OP50 diet supplemented with 64 nM vitamin B12 were synchronized using sodium-hydroxide–buffered sodium hypochlorite treatment. Synchronized L1 animals were cultured in specified conditions until L4 stage. At least 10 L4 animals per condition were imaged using Nikon A1 point-scanning confocal microscope with 561 nm laser. Imaging was performed using an Apo TIRF, N.A. 1.49, 60× oil immersion objective in galvano imaging mode.