Guinea pig anti insulin antibody

Human pancreatic islets of non-diabetic subjects were cultured with standard medium, or in the presence/absence of IGFBP3 and/or in the presence/absence of Ecto-TMEM219 for 72 h as previously described (see “Pancreatic islets and Interventional studies”). Islets were detached with Versene (ThermoFisher Scientific) and stained with the BD Horizon™ Fixable Viability Stain 510 (FVS510, BD Biosciences 564406, San Jose, CA), which ensures accurate assessment of cell viability in samples after fixation and/or permeabilization as per manufacturer’s instructions. After being stained with BD Horizon™ Fixable Viability Stain 510, cells were next fixed and permeabilized with Fixation and Permeabilization Solution Kit (554714, BD Biosciences, San Jose, CA)^{51 (link)} and finally stained with guinea pig anti-insulin antibody (1:200, Guinea Pig, ThermoFisher Scientific, PA1-26938) followed by AlexaFluor488 anti-guinea pig (1:200, ThermoFisher Scientific, A-11073). Flow cytometry analysis was performed using a BD FACS Celesta flow cytometry system (BD Biosciences) and analyzed using Flowjo software (Version 6 and Version 10, Tree Star, Ashland, OR).

Pancreatic tissue sections were fixed, rehydrated and permeabilized with 0.1% Triton X-100 for 2 min. The sections were washed with 0.1% Tween-20 PBS (PBS-T) containing Image-It FX signal enhancer (Thermo Fisher Scientific) for 1 h and incubated with primary antibodies overnight at 4 °C [guinea pig anti-insulin antibody (1:100, Thermo Fisher Scientific), MANF (1:100, Abnova), and Ki67 (1:100, Cell Signaling Technology)]. The tissue sections were washed three times in PBS-T and incubated with secondary antibodies for 1 h at room temperature. Images were obtained with a Zeiss LSM 5 PASCAL confocal microscope with LSM Image software.

For measurement of β-cell mass, every 40th pancreatic section was immunostained with guinea pig anti-insulin antibody (1:100, Thermo Fisher Scientific) and counterstained with hematoxylin. The β-cell mass for each mouse was quantified using Image Pro Plus software (Media Cybernetics, Rockville, MD) by obtaining the fraction of the cross-sectional area of pancreatic tissue (exocrine and endocrine) positive for insulin staining, and then multiplying this by the pancreatic weight.