Mouse anti nkx6

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.

Single cell dissociations were fixed in 4% paraformaldehyde and blocked with 5% normal donkey serum in 0.1% Triton-X PBS. Cells were incubated overnight with primary antibodies (CHGA, C-pep, NKX6.1, PDX1, OCT3/4), washed and subsequently incubated with appropriate secondary antibodies. Stained, fixed cells were analyzed by ZE5 cell analyzer (Bio-Rad). Primary and secondary antibodies used: mouse anti-OCT-3/4 (Santa Cruz Biotechnology, cat. no. sc-5279, clone C-10; RRID: AB_628051), chromogranin A Rabbit anti-Human, Invitrogen (catalog #: PIMA533052), rat anti-C-peptide (Developmental Studies Hybridoma Bank, University of Iowa, cat. no. GN-ID4-S), mouse anti-NKX6–1 (Developmental Studies Hybridoma Bank, University of Iowa, cat. no. F55A12-S), goat anti-PDX1 (R&D Systems, cat. no. AF2419), anti-rat AlexaFluor 488 (Invitrogen, cat. no. A21208), anti-mouse AlexaFluor 488 (Invitrogen, cat. no. A21202), anti-mouse AlexaFluor 647 (Invitrogen, cat. no. A31571), anti-goat AlexaFluor 647 (Invitrogen, cat. no. A21447), anti-rabbit AlexaFluor 647 (Invitrogen, cat. no. A31573).