A056401 2

Preparation and immunofluorescence of alginate beads were performed as previously described (Vethe et al., 2019b (link)). The pancreas from the mice were fixed for 2 h in 4% PFA, before dehydration using a sucrose gradient of 10, 20, and 30% and embedded in Tissue Tek OCT compound (Sakura JP). Sections of 10 μm were obtained with a cryotome (Leica CM 1950, Leica, DE) and added on SuperFrost Plus slides (Thermo Scientific). The immunofluorescence staining was performed following indications provided by the supplier. The following primary antibodies were used: mouse anti-insulin IgG1 (1/500, I2018, Sigma-Aldrich), guinea-pig anti-porcine insulin (1/400, A056401-2, Dako), mouse anti-porcine glucagon (1/1000, G2654, Sigma-Aldrich). The following secondary antibodies were used at dilution 1/500: goat anti-mouse IgG1 A488, goat anti-guinea-pig A488, goat anti-guinea-pig A546, goat anti-mouse IgG1 A546. DAPI (1/1000, D1306, Molecular Probes) was used to stain the nuclei. The samples were mounted in Prolong Diamond Antifade Mountant Media (P36970, Life technologies). Image acquisition and analysis was performed on Andor Dragonfly confocal microscope and Imaris 9.1.2 (Bitplane AG) as we have previously described (Vethe et al., 2019b (link)). We also performed manual counting on images acquired using a 40x immersion objective on Leica TCS SP5 confocal (Leica Microsystems CMS GmbH).

Similar to our previous report^{39 (link)}, mice were euthanized under anesthesia with CO₂ followed by cervical dislocation. The pancreas was immediately dissected, weighed, and fixed in 10% formalin on ice for 30 minutes. Pancreata were then washed in PBS and transferred through a series of solutions, beginning with 30% sucrose in PBS, 1:1 30% sucrose:OCT, and OCT before cryopreservation in OCT and storage at −80 °C. 10-micron sections, separated by 200 microns, were cut on positively charged slides. For each pancreas, one slide, containing 3 distinct sections, was post-fixed, quenched of peroxidase activity with 3% H₂O₂, and immunohistochemically labeled using guinea pig anti-insulin primary antibody (Dako A056401-2), diluted 1:500 in antibody diluent, and co-stained with hematoxylin (Sigma, GHS280). Slides were imaged using an automated pan-and-stich microscope at 10× (Evos). β-cell fractional area was determined by quantifying the percent of insulin-positive pancreas area as a total of the full pancreas area for each section, followed by averaging of 3 distinct sections per mouse. Images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD) with shading correction. β-cell mass was calculated by multiplying β-cell fractional area by the pancreatic wet weight.

Cells were cultured on glass coverslips and fixed in 2% paraformaldehyde (PFA) for 15 min. The immunofluorescence protocol performed conformed to indications provided by the supplier. The following primary antibodies were used: mouse anti-β-catenin (1/500, ab22656, Abcam), rabbit anti-BMP4 (1/500, ab39973, Abcam), rabbit anti-ROR2 (1/50, ab92379, Abcam), rabbit anti-c-JUN (1/500, ab31419, Abcam), mouse anti-insulin (1/500, I2018, Sigma-Aldrich), guinea-pig anti-porcine insulin (1/500, A056401-2, Dako), mouse anti-porcine glucagon (1/500, G2654, Sigma-Aldrich), rat anti-somatostatin (1/100, sc-47706, Santa Cruz), guinea-pig anti-PDX1 (1/500, ab47308, Abcam), and rabbit anti-NKX6.1 (1/100, NBP1-82553, Novus), rabbit anti-MAFA (1/200, ab98859, Abcam). The following secondary antibodies were used: donkey anti-rabbit A488, donkey anti-rabbit A546, goat anti-mouse A568, chicken anti-rat A647, and goat anti-guinea-pig A647 (1/500, Molecular Probes). The nuclei were stained with DAPI (D1306, Molecular Probes). The samples were mounted in Prolong Diamond Antifade Mountant Media (P36970, Life technologies) and were analyzed with Leica TCS SP5 STED CW confocal microscope. No specific feature of the original data was obscured, eliminated or misrepresented.