Guinea pig anti insulin

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).

Adjacent sections of frozen in vitro autoradiography were immunostained for insulin expression. Sections were fixed in acetone (4 °C) for 5 min, allowed to dry for 15 min, and then washed in PBS and Dako washing buffer. Donkey serum (3% in PBS) was added for blocking and incubated for 20 min at RT, followed by overnight incubation with guinea pig anti-insulin 1:400 (Fitzgerald, Acton, MA, USA). Cat No. 20-IP30) at 4 °C. The next day, the sections were first washed in PBS 2 × 5 min and 1 × Dako washing buffer. Alexa 488-conjugated donkey anti-guinea pig secondary antibody (1:300, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Code No. 706–545-148) were added to the sections for 50 min at RT. Afterwards, the sections were rinsed 3 × 5 min PBS and then incubated with DAPI for 10 min to achieve a background. Finally, the sections were washed 2 × PBS and mounted in fluorescence mounting medium on protective glass.