Insulin a0564

Islet graft-bearing kidneys, pancreases, and intestines fixed in 4% paraformaldehyde were embedded in optimal cutting temperature compound and frozen on dry ice. 6–10μm sections were made on a Leica CM3050 S (Leica Biosystems, Buffalo Grove, IL). Immunofluorescent staining was performed using standard methods. Briefly, sections were blocked in 5% BSA for 1hr then incubated with primary antibodies overnight at 4°C. Sections are washed 3 × 5 min before incubation with secondary antibodies for 2 hours at room temperature and washed 3 × 5 min again. Slide covers were secured with Hard-set Mounting Medium with DAPI (Santa Cruz Biotechnology, Dallas TX). Slides were imaged on an EVOS M5000 Cell Imaging System (ThermoFisher). Post-processing and color channel merging was performed in Fiji (http://fiji.sc/) (Schindelin et al., 2012 (link)). Primary antibodies (1:100–200): αCD3 (17A2) and αCD45 (30-F11) were purchased from BioLegend, insulin (a0564) from Dako (Carpinteria, CA). Secondary antibodies (1:200–500): CF-594 and CF-488A α-Guinea Pig were purchased from MilliporeSigma (St. Louis, MO), Alexa Fluor-594 and Alexa Fluor-488 α-Rat, and Alexa Fluor-594 α-Rabbit were purchased from BioLegend.