Rabbit anti glucagon

For whole mount immunofluorescent staining, larvae were fixed in 4% paraformaldehyde at 4°C overnight. Following fixation, the whole gut region was dissected out and blocked for 1 hour with PBS with 0.2% Triton (PBST) and 10% fetal bovine serum. For cryosections, fish were similarly fixed and the gut-intestine system was dissected. Tissues were then immersed in 30% sucrose/PBS, embedded in optimal cutting temperature (OCT) compound, frozen in liquid nitrogen, and sectioned in 10 µm thickness using a cryostat. Primary antibodies used in this study included: mouse anti-Nkx6.1 (Developmental Studies Hybridoma Bank, F55A12), 1: 100. Guinea pig anti-Insulin (Dako, A0564), 1: 500. Rabbit anti-Glucagon (Dako, A0565), 1:400. Rabbit anti-Somatostatin (Dako, A0566), 1:500. Rabbit anti-DsRed (Clontech, 642496), 1:100. Mouse anti-2F11 (Abcam, ab71286), 1:200. Primary antibodies were incubated at 4°C overnight. After washing with PBST, samples were incubated with secondary antibodies (Jackson Immunoresearch, 1:300) in blocking buffer. Fluorescent images were acquired with Nikon A1 scanning confocal microscope. Cell counting was carried out manually. Briefly, whole mount tissues were scanned by confocal microscope and maximum projections were assembled from Z-stacks. Cell numbers were counted from reconstructed images. Students t-test was implemented for statistical analysis.

Frozen/fixed mouse eyes bearing human islet grafts and recipient mouse pancreata were thawed at 4ºC, transferred into Sakura cassettes and fixation process was repeated with 10% formalin buffered solution for six hours at RT. Dehydration of eye tissue was performed in a VIP3000 after which eyes were embedded into paraffin and sectioned (4 μm) for immunofluorescence staining. Used antibodies included: guinea pig anti-insulin (Dako, diluted 1:35) and rabbit anti-glucagon (Dako, diluted 1:50), rabbit anti-Ki67 (Ventana).