RRID:AB_944318

AB_944318

RRID:AB_944318

RRID:AB_944318

RRID:AB_944318

RRID:AB_944318

Pancreas immunohistochemical and immunofluorescence analysisFFPE pancreatic sections (5 μm thickness) were analyzed as briefly follows. After deparaffinization and rehydration, sections were incubated with Tris-buffered saline (TBS, Sigma-Aldrich) supplemented with 3% H2O2 to block endogenous peroxidases (only for immunohistochemical experiments) and with TBS supplemented with 3% bovine serum albumin (BSA, Sigma-Aldrich) to reduce nonspecific reactions. Antigen retrieval was performed with 10 mM citrate buffer, pH 6.0.For immunohistochemical analysis, sections were incubated with rabbit polyclonal anti–human MPO (Abcam, ab45977) and swine polyclonal anti-rabbit IgG-HRP (DAKO, P0217) as secondary antibody. MPO signal was detected with 3,3′-diaminobenzidine (DAB Quanto, ThermoFisher Scientific, TA-060-HDX). Sections were then incubated with Mayer’s hematoxylin solution (Sigma-Aldrich) to counterstain nuclei, dehydrated, and mounted with Eukitt (Bio-Optica). Sections from the Siena cohort were also incubated with mouse monoclonal anti–human glucagon (R&D Systems, clone 181402, MAB1249) and goat anti-mouse IgG–alkaline phosphatase (ThermoFisher Scientific, 31320) as secondary antibody. Glucagon signal was detected with Liquid Fast Red (ThermoFisher Scientific, TA-060-AL) supplemented with levamisole endogenous alkaline phosphatase inhibitor (DAKO, Agilent Technologies, X3021).For immunofluorescence analysis, sections were incubated with rabbit polyclonal anti–human MPO (Abcam) and mouse anti–citrullinated histone H3 (anti-CitH3; citrulline R2 + R8 + R17, clone 7C10, LifeSpan Biosciences, LS-C144555) antibodies and with goat anti-rabbit IgG Alexa Fluor 488 (Molecular Probes, ThermoFisher Scientific, A11034) and goat anti-mouse IgG Alexa Fluor 594 (Molecular Probes, ThermoFisher Scientific, A11032) as secondary antibodies. nPOD sections were also stained with polyclonal guinea pig anti-glucagon antibody (LifeSpan Biosciences, LS-{"type":"entrez-nucleotide","attrs":{"text":"C20275","term_id":"1632546","term_text":"C20275"}}C20275) and goat anti-guinea pig IgG Alexa Fluor 647 (Molecular Probes, ThermoFisher Scientific, A21450) as secondary antibody. DNA was counterstained with Hoechst 33342 (ThermoFisher Scientific, 62249). Finally, sections were mounted using Vectashield (Vector Laboratories, H1000) mounting medium.OCT-embedded tissues (5 μm thickness) were methanol/acetone (1:1, –20°C) fixed, then blocked (PBS supplemented with 1% denatured BSA) and incubated with primary antibodies: mouse monoclonal anti-MPO (Bio-Rad, clone 4A4, 0400-0002) and polyclonal rabbit anti-CitH3 (citrulline R2 + R8 + R17, Abcam, ab5103). Sections were washed and incubated with the proper secondary antibodies: goat anti-mouse IgG (H+L) Alexa Fluor 488 (Jackson ImmunoResearch, 115-545-003) and goat anti-rabbit IgG (H+L) Alexa Fluor 546 (ThermoFisher Scientific, A11035). DNA was counterstained with Hoechst 33342. Sections were mounted on slides with homemade Mowiol mounting medium (glycerol, G5516; Mowiol 4-88, 81381; and Dabco 33-LV, 290734, Sigma-Aldrich).

RRID:AB_944318