Alexa fluor 488 donkey anti guinea pig

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).

Human islets were trypsinized so that a cell suspension was obtained, and then transferred to a clearly defined area of poly-lysine glass slides using a Cytospin 4 centrifuge (Thermo Scientific, Waltham, MA, USA). Thereafter cells were fixed with 4% (w/v) paraformaldehyde for 5–10 min at room temperature, blocked for 30 min using 2% BSA, permeabilized with 0.1% Tris-NaCl for 10 min, and stained overnight with the following primary antibodies: rabbit anti-GDF15 (Abcam, 1:250), guinea pig anti-insulin (Fitzgerald, Sudbury, MA, USA, 1:250), and mouse anti-glucagon (Thermo Fischer Scientific, 1:1000). All slides were washed with phosphate-buffered saline (PBS) and, prior to counterstaining, with diluted secondary antibodies: ALEXA Fluor 594 donkey anti-rabbit (Jackson, bar Harbor, ME, USA, 1:250), ALEXA Fluor 488 donkey anti-guinea pig (Jackson, 1:250), and ALEXA Fluor 594 goat anti-mouse (Life technologies, Carlsbad, CA, USA, 1:250) for 1 h at room temperature. Cells were washed with PBS and then mounted with Biotium’s (Freemont, CA, USA) EverBrite™ mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI) for nuclei staining. Images were taken and analyzed with a Nikon Eclipse C1/TE2000-U microscopy (Nikon, Konan, Tokyo, Japan).

Retinal dissection was performed following established protocols in the Frankfort lab (Frankfort et al., 2013 (link); Tao et al., 2020b (link)). Dissected whole-mount retinas were fixed with 4% paraformaldehyde for 1 h at room temperature and blocked with 10% donkey serum overnight. Retinas were then incubated in primary antibodies [Collagen IV (EMD Millipore cat#AB756p; 1:300), CD31 (BD bioscience cat#550274; 1:50), and RBPMS (Phospho solutions cat#1832; 1:250)] diluted with 3% donkey serum for 5 days at 4°C, followed by overnight incubation at 4°C in secondary antibodies [Alexa fluor 647 donkey anti-rabbit (Jackson Immuno Research Labs cat# 711-605-152; 1:300), Cy3 donkey anti-rat (Jackson Immuno Research Labs cat#712-165-153; 1:300), Alexa fluor 488 donkey anti-guinea pig (Jackson Immuno Research Labs cat#706-545-148; 1:300), and Hoechst 33,342 nuclear staining (Invitrogen cat#H3570; 1:1,000)] diluted with 3% donkey serum.