Guinea pig anti insulin

Tissue sections were dewaxed in xylene and hydrated in serial dilutions of ethanol (2×; 100%, 90% and 70%) followed by water. Heat- and citrate-based antigen retrieval was performed using Vector Laboratories (CA, USA) antigen unmasking solution (H-3300) for 20 min and then the slides were allowed to cool to room temperature for 1 h. Slides were blocked with Dako blocking buffer (Agilent Technologies, CA, USA, #X0909) prior to incubation with antibodies in Dako antibody diluent (Agilent Technologies #S3022). Antibodies and the corresponding dilutions used are listed as follows: Developmental Studies Hybridoma Bank (DSHB, Iowa, USA) mouse anti-Proinsulin (GS-9A8; 1:50), rat anti-lysosomal-associated membrane protein 1 (LAMP1) (DSHB 1D4B; 1:50), rabbit anti-LC3A/B (Cell Signaling Technology, MA, USA, #12741; 1:100), rabbit anti-p62 (Abcam, Cambridge, UK, #ab91526; 1:200) and guinea pig anti-insulin (BioRad, CA, USA, #5330-0104G; 1:500). Highly cross-adsorbed fluorescently conjugated secondary antibodies were used. Images were collected on either a Zeiss (Carl Zeiss, Oberkochen, Germany) LSM 700 confocal microscope using a 63X/1.4 numerical aperture oil objective, or a Zeiss LSM 800 confocal microscope equipped with Airyscan using a 63X/1.4 numerical aperture oil objective.

Pancreas sections from human donors and human IAPP (hIAPP) transgenic mice were stained for macrophages using CD68/SR-D1 antibody (Novus Biologicals, Littleton, CO, USA), or using guinea pig anti-insulin (DAKO) mouse anti-C4BP, sheep anti-pancreatic polypeptide (Bio-Rad) or Congo Red. See ESM Methods for further details.