Z fix

Tissue designated for IHC analysis was fixed overnight in Z-Fix (NC9050753, Thermo Fisher Scientific), washed with PBS for 30 minutes at RT, processed and embedded in paraffin. The use of human samples was approved by the Henry Ford Health Institutional Review Board. IHC single- and multiplex-staining were performed on 4 μm sections using the Discovery ULTRA stainer (Roche). Antibodies and dilutions are provided in Supplementary Table 1. Hematoxylin and Eosin (H&E) and Picrosirius Red staining were performed according to standard protocols. Stained sections were scanned with the PANNORAMIC SCAN II (3DHistech Ltd.) and the number of positively stained cells / area was quantified with HALO software v3.5.3577.214 (Indica Labs). Representative images were taken from scanned images or with the Stellaris 5 confocal microscope (Leica).

Isolated islets were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 15 min and permeabilized with 0.3% Triton X-100 in PBS (PBST) for 10 min at room temperature. After incubation with blocking buffer PBS with 10% normal goat serum for 1 h at room temperature, islets were incubated overnight at 4 °C with primary antibodies diluted in PBST. The next day, islets were washed, incubated with secondary antibodies for 1 h at room temperature, and washed again in PBST. DAPI provided nuclear counterstain. Islets were mounted on glass slides with Prolong Gold Anti-fade (Thermo Fisher P36930). Pancreas sections were either flash frozen in optimal cutting temperature (OCT) compound and cut into cryosections or fixed in zinc formalin (Z-Fix, Thermo Fisher NC9351419) or 4% PFA (Electron Microscopy Sciences 15710), dehydrated in 10 to 30% sucrose, and prepared as paraffin or frozen sections. OCT sections were fixed in methanol and stained using the above methods. All islet and OCT sections were imaged using an inverted Zeiss LSM880 fluorescence microscope (Nikon, Ti-E).