Mab13361

For immunofluorescence staining, frozen tissue sections were cut at 7-8 μm, air dried and permeabilized with ice-cold methanol for 15 min. Sections were briefly washed twice with PBS and placed in 0.3% Triton X-100 10 min before blocking with 10% KPL (5140-0011, SeraCare) in PBS for 1 h at room temperature. The sections were incubated with primary antibodies against phospho-Stat3 (1:200; 9145, Cell Signaling Technology), insulin (1:200; PA1-26938, ThermoFisher), proinsulin (1:200; MAB-13361, R&D), Reelin (1:1000; AF3820, R&D), and GFAP (1:200; LS-B4775, LifeSpan Bioscience) at 4 °C overnight. Sections were washed three times with PBS and incubated for 1 h in the dark with the corresponding fluorophore-conjugated secondary antibodies, Alexa Fluor 488 Chicken anti-rabbit IgG (1:500; A-21441, Invitrogen), Alexa Fluor 488 Goat anti-guinea pig IgG (1:1000; A-11073, Invitrogen), Alexa Fluor 555 Goat anti-rabbit IgG (1:1000; A-32732, Invitrogen), Alexa Fluor 555 Goat anti-chicken IgG (1:500; A-21437, Invitrogen), Alexa Fluor 647 Goat anti-mouse IgG (1:500; A-21235, Invitrogen). Sections were counterstained using DAPI (1:1000 from 1 mg/ml; D9542; Sigma) to visualize cell nuclei and cover slipped with Fluoroshield (Sigma, F6182). Images were captured using the Olympus confocal microscope FV3000. ImageJ software was used to quantify MFI per islet area in the Il22ra1 x Ins2-cre animals.