Polyclonal guinea pig anti insulin

Paraffin-embedded sections close to the middle of the pancreata (representing the largest pancreatic area of the pancreas) were cut at 100 μm apart at 2 different levels. Sections were immunostained with polyclonal guinea pig anti-insulin (1:200) (Dako) overnight at 4°C. Immunofluorescence staining was carried out with Alexa Fluor 647 goat anti–guinea pig (1:200; Invitrogen, Thermo Fisher Scientific) secondary antibody for 1 hour incubation at room temperature and subsequently stained with 0.5% thioflavin S (MilliporeSigma) for 5 minutes. Images were obtained using a Zeiss confocal microscope or Zeiss Axioscan slide scanner using Zen software. All quantifications were performed by HALO version 2.0.1145.14 (Indica Labs). Amyloid severity was measured as total amyloid area per total islet area, and amyloid prevalence was analyzed by total number of islets positive for thioflavin S detected as a percentage of the total number of islets. β Cell area was quantified as total islet insulin-positive staining per total islet area. More than 50 islets per sample were analyzed.

Pancreata from WT and BKO mice fed two weeks on CD and HFD were dissected, fixed in 10% formalin neutral buffered solution and paraffin embedded. Three-micrometer-thick pancreatic sections from 3 different levels (>150 μm apart) for each pancreas were deparaffinized, rehydrated, boiled in citrated buffer (10 mM; pH = 6.0) only in case of Ki67 staining, permeabilized with 1% Triton X-100 (Sigma–Aldrich) in Dulbecco's phosphate buffer saline (PBS; Sigma–Aldrich) and blocked with 5% Donkey serum in PBS. Samples were incubated overnight with polyclonal guinea pig anti-insulin (1:500; Dako, Glostrup, Denmark) and mouse-anti glucagon (1:1000; Sigma–Aldrich) or rabbit anti-Ki67 (1:200; Thermo Fisher Scientific, Waltham, MA, USA). As secondary antibodies, Alexa Fluor 488 anti-guinea pig (1:250; Jackson I.R., Newmarket, UK), 555 anti-mouse (1:250; Invitrogen, Carlsbad, CA, USA) and 455 anti-rabbit (1:400, Dako) were used. Hoechst 33342 (Sigma Aldrich) was used to stain nuclei. Images were taken with Leica TCS SPE confocal microscope and the analysis performed using Fiji (ImageJ) version 2.1 [28 (link)]. Total β-cell mass was calculated by multiplying their fractional area per pancreas weight. The percentage of proliferating β-cells was calculated as the percentage (%) of Ki67-positive β-cells with respect to total β-cells.

INS(832/13) and islet cells were seeded in 8-well chambers (Nalgene Nunc, Thermo Fisher). After 48 h, cells were either left untreated (NT cells) or infected with E16. Cells treated with LG were included as a positive control for autophagy. Thereafter, LC3II-GFP (1:400) (Premo Autophagy sensors BacMam 2.0, Life Technologies, OR, USA) was introduced for 18 h. Two hours prior to the end of the incubation period (i.e. at 16 h), LysoTracker Red DND-99 (1:2000) (Life Technologies) was added. LysoTracker Red stains lysosomes and autolysosomes. Islet cells were counterstained with polyclonal guinea pig anti-insulin (1:500) (DAKO, Jena, Germany) and secondary antibody Alexa Flour 594 anti-guinea pig IgG (H+L) conjugate (Thermo Fisher). Cells were washed twice in PBS, pH 7.4, fixed with 4% paraformaldehyde, washed twice again in PBS and mounted in VECTASHIELD Mounting Medium with DAPI (nuclear staining).