The pancreas was removed from a centrifuge tube with 4% paraformaldehyde and then a small part of the complete pancreas was cut. The pancreas tissues were soaked in 15% sucrose and dehydrated overnight. Then tissues were transferred into 30% sucrose and dehydration overnight. The dehydrated tissue was taken out and embedded in OCT Compound (SAKURA, 4583) and frozen in −80 °C. The tissues were subsequently sliced into 8 μm sections using freezing microtome (LEICA CM1850UN).
Sections were fixed with 4% paraformaldehyde and permeabilized for 20 min. After twice washes with PBS for 5 min, permeabilization with 0.5% Triton X-100/SDS/PBS for 20 min at room temperature, followed by three washed in PBS. Slides were blocked in 5%FBS/PBS/1% BSA for 3 h at room temperature and incubated in the indicated antibodies at 4 °C overnight. After twice washed with PBST for 5 min and washed with PBS for 5 min, dark incubated slides with FITC-labeled secondary antibody (Bioss, bs-0358G) for 2 h at room temperature. Slides were then washed twice with PBST for 5 min and washed with PBS for 5 min, followed were incubated with Hoechest 33342 for 10 min at room temperature. After three washes with PBS, the slides were covered with fluorescence decay resistant medium (BOSTER, AR1109). All slides were quantified by fluorescence microscopy.
Sections were fixed with 4% paraformaldehyde and permeabilized for 20 min. After twice washes with PBS for 5 min, permeabilization with 0.5% Triton X-100/SDS/PBS for 20 min at room temperature, followed by three washed in PBS. Slides were blocked in 5%FBS/PBS/1% BSA for 3 h at room temperature and incubated in the indicated antibodies at 4 °C overnight. After twice washed with PBST for 5 min and washed with PBS for 5 min, dark incubated slides with FITC-labeled secondary antibody (Bioss, bs-0358G) for 2 h at room temperature. Slides were then washed twice with PBST for 5 min and washed with PBS for 5 min, followed were incubated with Hoechest 33342 for 10 min at room temperature. After three washes with PBS, the slides were covered with fluorescence decay resistant medium (BOSTER, AR1109). All slides were quantified by fluorescence microscopy.