Bz x viewer software

Tiling and stitching were performed with Keyence BZ-X Viewer software. Image quantification was performed in ImageJ as shown in fig. S23. Images were split into component channels. Nuclei (blue channel) were quantified by applying Gaussian Blur (3 Sigma), thresholding, performing watershed calculation for segmentation, and counting. Lipid accumulation was quantified by applying Gaussian Blur (2 Sigma), thresholding, and quantifying the total area above the threshold. The level of adipogenesis, expressed as the adipogenic index, was assessed by dividing the total lipid area by the total number of nuclei to obtain a number with arbitrary units for comparing absolute levels of adipogenesis. Relative adipogenesis was calculated as the ratio of the adipogenic indices in treatment and control samples; here, the control is the untreated sample from the same cell type and BR (for example, ICAM1–TGFβ BR A/ICAM1 control BR A). This ratio represents induction or suppression compared with the baseline.

Images for histological confirmation were taken with the BZ-X710 all-in-one fluorescence microscope with the BZ-X viewer software under a 10X, 0.45 NA objective (Keyence). Images for the monosynaptic Rabies tracing were taken at the Salk Institute Waitt Advanced Biophotonics Core with the Olympus VS-120 Virtual Slide Scanning Microscope under an Olympus UPLSAPO 4X, 0.16 NA objective. Images for MOR immunohistochemistry, SypGFP terminals in the preBötC, projection mapping, and RNAscope were taken by an FV3000 Confocal Laser Scanning Microscope with FV31S-SW software under Olympus UPLSAPO 4X, 0.16 NA; 10X, 0.40 NA; 20X, 0.75 NA; 40X, 1.30 NA, or 60X, 1.42 NA objectives (Olympus). For quantification, images were processed with the same gain, offset, and exposure time.

Mouse pancreas was fixed in 10% neutral buffered formalin and embedded in paraffin for sectioning. IHC and IF were performed as previously described^{33 (link)}. Antibodies used are listed in Supplementary Table 2.. Some slides, as indicated, also included nuclear staining by DAPI (4’, 6-diamidino-2-phenylindole). Ki67 IHC was used to calculate Ki67⁺ cells against total cell number from >4 views in each mouse. Co-IF of PDX1 and insulin was used to calculate the frequency of PDX1⁺ cells among insulin⁺ cells from 3–5 representative islets for each mouse. Co-IF of cleaved caspase-3 (Asp175) and insulin was used to calculate the frequency of apoptotic cells among insulin⁺ cells from 3–5 representative islets for each mouse. H&E staining was conducted by following the manufacturer’s instructions (ab245880, Abcam). Images were taken using a BZ-X700 microscope (Keyence) and BZ-X viewer software (Keyence, version 1.0.0). Islet area was measured using ImageJ and average islet area was calculated from more than 8 representative islets for each mouse.

Adipogenesis was assessed by staining with Bodipy 493/503 (Invitrogen catalog no. D3922) for lipid accumulation and Hoechst 33342 (Thermo Fisher catalog no. 62249) for nuclei³² . Briefly, cells were differentiated in 384 well tissue culture plates (Sigma-Aldrich catalog no. CLS3770), fixed with 4% paraformaldehyde, stained, and imaged on a Keyence inverted microscope (BZX-710) using Keyence BZ-X Viewer Software (1.3.1.1) with the following filters: DAPI (ex, 360/40 nm; em, 460/50 nm; Keyence, OP-87762) and GFP (ex, 470/40 nm; em, 525/50 nm; Keyence, OP-87763) filters. Images were acquired at 20x in a 7×7 tiled grid and stitched to capture the entirety of each well. Tiling and stitching were performed with Keyence BZ-X Analyzer software (1.3.0.3). Image quantification was performed automatically in ImageJ (version 1.52E) using a macro which: 1) Split images into component channels, 2a) for the nuclei channel applied a 3-Sigma Gaussian blur, performed thresholding to identify signal above background, performed watershed to segmentation, and counted the number of nuclei, 2b) for the lipid channel applied a 2-Sigma Gaussian blurm performed thresholding to identify signal above background, and counted the area (#of pixels) with signal above threshold. The amount of adipogenesis was calculated as Lipid Area/#nuclei.