Insulin

Samples were fixed in 4% buffered formaldehyde for 24 h, washed in PBS, transferred into 70% EtOH in ddH₂O, and then embedded in paraffin and sectioned (5mm thickness) using a Leica RM2255 rotary microtome, by the Brain Tumor Research Center (BTRC) Histology Core, or by the Biobank and Tissue Technology Lab at UCSF. Hematoxylin and eosin staining was performed using standard methods. Stained slides were imaged using an Aperio AT2 slide scanner and data were processed using QuPath software. Total cell counts, Ki67 stains in all tissues, and cleaved caspase-3 stains in RT2 tumors were automated in a blinded fashion; cleaved caspase-3 stains in INS-1 xenografts were quantified manually in a blinded fashion. Antibodies used for immunohistochemistry: BiP [C50B12] (Cell Signaling Technology #3177, 1:200), CD31 (CST #77699 1:100), Chromogranin A [polyclonal] (Cell Marque, 1:4), Cleaved Caspase-3 (CST #9661 1:200), Insulin (DAKO A0564, 1:200), IRE1α (CST #3294, 1:100), Ki67 (Ventana #790–4286, Undiluted), Myc (Sigma M4439, 1:5000), Synaptophysin [LK2H10 clone] (Cell Marque, 1:100).

Pancreata were fixed with 4% PFA at room temperature for 4 h, paraffin embedded, and immunostaining was performed as previously described (Evans-Molina et al., 2009 (link)) using the following antibodies: insulin (Santa Cruz) and counter stained with peroxidase conjugated anti-rabbit IgG (Vector). Insulitis was scored from immunohistochemical staining as described previously (Tersey et al., 2014 (link)) using at least 3 pancreas sections 70 μm apart from 5 mice per group and the images were acquired using an EVOIS XL Core microscope (Life Technologies). For studies using immunofluorescence we used the following primary antibodies: insulin (Dako; 1:400), GDF15 (Bioss; 1:200), SPP1 (Thermo Fisher Scientific; 1:200), 4-HNE (Abcam; 1:200), glucagon (Abcam; 1:300) and counter stained with following secondary antibodies: goat anti-guinea pig (Alexa 488; 1:400), donkey anti-rabbit (Alexa-568; 1:250) and donkey anti-mouse (Alexa-647; 1:300). The images were acquired using LSM 800 confocal microscope (Carl Ziess, Germany). Experiments without the primary antibody and with a competition assay by adding recombinant protein were run in parallel to assess the specificity of the staining.

Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin and 5 μm sections were prepared. Routine H&E and immunohistochemistry (IHC) were performed as previously described (Hingorani et. al., 2003 (link)) and IHC scoring was done blinded. Biotinylated secondary antibody and the Elite vectastain kit (Vector Lab, CA, USA) were used for signal detection and DAB was used as a chromogen substrate. Primary antibodies: amylase (1:800, Sigma, Chicago, IL, USA); CK-19 (DakoCytomation, Denmark); cleaved caspase 3 (1:100, Biocare, CA, USA); Dpc4 (1:300, Abcam, MA, USA); insulin (1:200, Dako, CA, USA); Ki-67 (1:25, Dako, CA, USA); and Runx3 (1:200 for murine and 1:100 for human IHC, Cell Signaling, MA, USA).