Mouse anti human scgf clec11a

Total protein was collected from human islets and EndoC-βH1 cells using Pierce RIPA Buffer (Thermo Fisher) and Protease Inhibitor Cocktail (Sigma Aldrich). The protein was measured using Bio-Rad DC Protein Assay (Bio-Rad). An equal amount of protein was separated by Mini-PROTEAN TGX Stain-Free Gels (Bio-Rad) and transferred to a PVDF membrane. After blocking with 5% non-fat milk, the membrane was incubated with diluted primary antibodies overnight at 4°C. Primary antibodies used for western blot were as follows: mouse anti-human SCGF/CLEC11A (1:500) (R&D Systems), rabbit anti-human ITGA11 (1:500) (LSBio, Shirley, MA, USA), rabbit anti-human PDX1 (1:3000) (Abcam), and rabbit anti-beta-actin (1:1000) (Cell Signaling Technology). After washing and incubation with a horseradish peroxidase (HRP)-labelled secondary goat anti-rabbit antibody (1:1000) (Thermo Fisher) for 60 min, reactivities were detected by an HRP chemiluminescent substrate kit (Bio-Rad). Positive signals were quantified using Image J software. For immunofluorescence western blot, the immunodetection was performed using IRDye 800CW- (green) and IRDye 680 RD (red)-labelled secondary antibodies diluted 1:15 000, respectively (LI-COR, Lincoln, NE, USA), and fluorescence was recorded and analysed in the Odyssey FC instrument (LI-COR).

For the co-localization experiment of CLEC11A with insulin or CLEC11A with glucagon, immunolabelling was performed on partially digested human islets. Briefly, islets were trypsin treated for 5–8 min, and smaller cell aggregates were cytospinned (175 g for 3 min) onto polylysine pre-coated slides, followed by fixation in 4% PFA for 5 min. After washing with PBS three times, the cells were permeabilized in 0.1% Triton X-100 for 10 min, followed by blocking in 2% fetal calf serum (FCS) for 60 min and then incubated for 60 min at 37°C with the primary antibodies. Following washing with PBS twice and then Dako washing buffer (Agilent) once, all slides were incubated with diluted secondary antibodies for 1 h at room temperature. Cells were then washed four times with PBS and mounted with VECTASHIELD Hard Set mounting medium with DAPI (Vector Laboratories, Newark, NJ, USA). Images were acquired using confocal microscopy (Nikon).
Primary antibodies were mouse anti-human SCGF/CLEC11A (1:300) (R&D Systems), guinea pig anti-human insulin (1:300) (Fitzgerald, Acton, MA, USA), and goat anti-human glucagon (1:300) (Abbexa, Cambridge, UK). Secondary antibodies were Alexa Fluor 594 donkey anti-mouse (1:300) (Jackson ImmunoResearch), Alexa Fluor 488 donkey anti-guinea pig (1:300) (Jackson ImmunoResearch), and Alexa Fluor 488 rabbit anti-goat (20 μg/mL) (Invitrogen).