G2654

To profile endocrine cell types after sorting, both sorted and unsorted islet cells were seeded in Matrigel-coated flat-bottom 96-well plates (1000 cells/200 μL/well) immediately after FACS. Next day (day 1), the cells were fixed in 4% PFA (Wako) for 15 min at room temperature and washed with PBS. Cells were then permeabilized and blocked with PBS containing 0.25% Triton-X and 5% donkey serum (Sigma) for 1 hr at room temperature. Primary antibodies C-peptide (DSHB, 1:200) and glucagon (MilliporeSigma, G2654, 1:10,000) diluted in antibody dilution buffer (Abcam) were added to the wells for overnight at 4°C. Cells were washed three times with PBS and the secondary antibody, diluted in PBS, was added to the wells for 1 hr at room temperature. Cells were washed three times with PBS and DAPI (Sigma) was added to the wells. Images were captured using an Olympus IX51 Inverted Microscope. For estimation of cell composition, ~4000 cells were counted per donor, and data were expressed as percentage of hormone+ cells.

These antibodies were used in this study for immunohistochemistry; Primary antibodies were goat anti-WNT4 (R&D system, AF475, 1:100) (Fig. 1g), rabbit anti-WNT4 (Bioss Antibodies, BS-6134R, 1:100)(Supplementary Fig. 3), guinea pig anti-Insulin (DAKO, A0564, 1:100), mouse anti-Glucagon (Sigma, G2654, 1:800), chicken anti-GFP (Abcam, ab13970, 1:1000), rabbit anti-Ki67 (Abcam, ab16667, 1:100), mouse anti-active beta-catenin (Millipore, 05-665, 1:100), rabbit anti-pMLC (Cell Signaling Technology, 3674, 1:100). Secondary antibodies were donkey anti-mouse Alexa Fluor^TM 568 (Thermo Fisher, A10037, 1:1000), donkey anti-mouse Alexa Fluor® 647 (Jackson ImmunoResearch Europe Ltd, 715-605-150, 1:800), donkey anti-chicken Alexa Fluor® 488 (Jackson ImmunoResearch Europe Ltd, 703-545-155, 1:800), goat anti-chicken Alexa Fluor^TM 488 (Thermo Fisher, A-11039, 1:1000), donkey anti-goat Alexa Fluor® 488 (Abcam, ab150129, 1:1000), donkey anti-guinea pig Texas red (Abcam, ab6906, 1:300), donkey anti-guinea pig Biotin (Jackson ImmunoResearch Europe Ltd, 706-065-1480, 1:400), donkey anti-rabbit Biotin (Jackson ImmunoResearch Europe Ltd, 711-065-152, 1:200), Streptavidin Alexa Fluor® 647 (Jackson ImmunoResearch Europe Ltd, 016-600-084, 1:1000). Nuclei were stained with DAPI (Sigma, D9542-1MG, 1:10000).

Larvae were harvested at 7 dpf, fixed for 1 to 2 h at room temperature in 4% paraformaldehyde (PFA)/1% DMSO in PBS, then washed three times for 5 min each with 1× PBS/0.1% Tween 20. To improve access of antibodies to internal structures, the ventral skin was cut open. Larvae were incubated in blocking buffer (1% DMSO, 1% sheep serum, 1% BSA, 1% Triton X-100 in 1× PBS) for at least 60 min at room temperature. Samples were then incubated overnight at 4°C with primary antibody, washed and then reblocked and incubated in secondary antibody overnight at 4°C. Primary antibodies were: mouse anti-GFP (Roche, 11814460001, 1:100), mouse anti-glucagon (Sigma, G2654, 1:100), mouse anti-2F11 (Abcam, ab71286, 1:100), rabbit anti-somatostatin (Dako, A0566, 1:200), guinea pig anti-insulin (Dako, A0564, 1:200), rabbit anti-GFP (antikoerper, ABIN110592, 1:200) and rabbit anti-dsRed (Clontech, 632496, 1:200).
Secondary antibodies were labeled with Alexa Fluor (Invitrogen) and diluted 1:1000. Nuclei were labeled by incubation overnight at 4°C in 100 ng/ml DAPI/1% DMSO in PBS. Phalloidin staining (Alexa Fluor 488 Phalloidin, ThermoFisher, A12379) was performed as described (Goody et al., 2012 (link)).