Goat anti nkx6

At the end of the culture period, islets were harvested, washed with PBS, and fixed in 4% paraformaldehyde (PFA) for 24 hours. PFA was removed and the islets were embedded in 1.5% agarose, subsequently embedded in paraffin, and sections made at 4 μm. After deparaffinization and rehydration, antigen retrieval was performed by microwaving slides in sodium citrate (0.01 M, pH 6.0). Sections were blocked with 5% donkey serum for one hour and incubated in primary antibody overnight at 4° C. Secondary antibodies were applied for 1 hour and nuclei were subsequently stained with DAPI (VECTASHIELD, Vector Laboratories). Slides were visualized with an Axio Imager (Carl Zeiss, Oberkochen, Germany). Overlay and cell counting were performed with ImageJ software (available at http://rsb.info.nih.gov/ij). Primary antibodies utilized in this study included: Guinea Pig anti-insulin, mouse anti-Ki67 (Dako), mouse anti-glucagon (Sigma), rabbit anti-Ki67, goat anti-PDX1 (Abcam), and goat anti-NKX6.1 (R&D). Secondary antibodies included: donkey Alexa Fluor-488 and -594 conjugates (Life Technologies and Jackson ImmunoResearch).