Anti fading mounting medium

The collected cells were fixed in 4% paraformaldehyde for 1 h, then the cells were spread onto poly-L-lysine coated microscope slides and air-dry. After three washings with PBS (5 min each) cells were incubated with 2% (v/v) Triton X-100 in PBS for 1 h at RT. Then, after three washes with PBS, the cells were blocked with 1% (wt/v) BSA and 1% goat serum in PBS for 30 min at RT, then incubation with primary antibodies diluted in blocking solution overnight at 4°C. The information for the primary antibodies (Abs) is present in Supplementary Table 2. The following morning, after three washes with PBS Tween 20 (0.5%) the slides were incubated with Alexa Fluor 546 goat anti-rabbit IgG (1:200) for 30 min in darkness at RT. The negative controls samples were incubated with secondary antibody and without primary antibody. Slides were washed with PBS Tween-20 three times and then incubated with DAPI (4.6-diamidino-2-phenylindole hydrochloride, 100 ng/ml) as nuclear stain for 5 min. After brief wash with ddH₂O, the slides were covered with an anti-fading mounting medium (Vector, Burlingame, USA). Fluorescent images were obtained with Leica Laser Scanning Confocal Microscope (LEICA TCS SP5 II, Germany) [118 (link)].

Pancreas was dissected, fixed in 4% freshly prepared paraformaldehyde (pH 7.4) for 24 h at 4 °C, and then processed routinely for paraffin embedding^{23 (link)}. For miRNA in-situ hybridization, sections were first deparaffinized and rehydrated, then treated with Proteinase K (Roch, 40 µg/ml) as described^{25 (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.