Pancreas was dissected, fixed in 4% freshly prepared paraformaldehyde (pH 7.4) for 24 h at 4 °C, and then processed routinely for paraffin embedding23 (link). For miRNA in-situ hybridization, sections were first deparaffinized and rehydrated, then treated with Proteinase K (Roch, 40 µg/ml) as described25 (link). Briefly, a total of 3 pmol of DIG-labeled Locked Nucleic Acid (LNA) probe for miR-30d (Exiqon) were mixed with 200 µl of hybridization buffer and applied onto the slides in order to hybridize at 37 °C overnight. Slides were then washed using 2X SSC (saline-sodium citrate) solution and incubated with alkaline phosphatase-conjugated sheep anti-DIG antibody (Roche) at 4 °C overnight. Alkaline phosphatase reaction was carried out with 50 mg/ml of nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) staining solution at room temperature overnight.
For immunohistochemistry, sections were immunostained with anti-insulin (Sigma), anti-glucagon (Sigma), or anti-GFP (Cell signaling) for overnight incubation at 4 °C. The immunodetection was processed with Alexa Fluor 488- or Alexa Fluor 596-conjugated secondary antibodies (Invitrogen) for 2 h at room temperature. Slides were then mounted with anti-fading mounting medium (Vector Labs) and the images were captured on Olympus FluoView FV1000 confocal microscopy or Leica fluorescence microscopy.
For immunohistochemistry, sections were immunostained with anti-insulin (Sigma), anti-glucagon (Sigma), or anti-GFP (Cell signaling) for overnight incubation at 4 °C. The immunodetection was processed with Alexa Fluor 488- or Alexa Fluor 596-conjugated secondary antibodies (Invitrogen) for 2 h at room temperature. Slides were then mounted with anti-fading mounting medium (Vector Labs) and the images were captured on Olympus FluoView FV1000 confocal microscopy or Leica fluorescence microscopy.