Immunostaining of Mouse Pancreatic Tissue

Mouse pancreas was collected and fixed in 4% formaldehyde at 4°C overnight, followed by paraffin embedding. Five-micron thick slides were cut and subjected to immunostaining. Slides were heated in 10mM sodium citrate, followed by blocking with donkey serum and incubated with various primary antibodies: Ki67 (#550609, BD, USA), Anti-METTL3 (#195352, Abcam, USA), Anti-METTL14 (#HPA038002, Sigma, USA), ALKBH5 (#HPA007196, Sigma, USA), PDX1 (#5679, Cell Signaling, USA), Insulin (#ab7842, abcam, USA), Glucagon (#G2654, Sigma, USA), Somatostatin (#ab64053, abcam, USA). Specific signal was detected by using fluorescence-conjugated secondary antibodies (Jackson Immunoresearch, Alexa 488, Alexa 594, and AMCA) (please see Reporting Summary for details on antibodies used). Images were captured using Zeiss Axio Imager A2 upright fluorescence microscope. The β-cell mass was calculated by generating the ratio of the cross-sectional area of total number of pixels of Insulin positive cells to the cross-sectional area of total number of pixels of the pancreatic tissue, multiplied by the pancreas weight of the mouse. We evaluated β-cell proliferation by co-immunostaining one section of each pancreas sample with Ki67, Insulin and DAPI and counting 1000–2000 cells in a blinded manner by a single observer^{33 (link),34 (link),37 (link)}.