Confocal laser scanning microscope sp8 inverted confocal microscope

Pancreas tissue was cut into 5 µm sections and deparaffinized with xylene for 7 min (3 times). The slides were gradually rehydrated in various concentrations of alcohol and washed with deionized water. After washing with PBS, the slides were treated with a methanolic solution of 3% of H₂O₂ for 20 min at room temperature to quench endogenous peroxide activity. To expose the antigen, the pancreatic sections were boiled in the target repair solution (0.01 m sodium citrate, pH 6.0) for 10 min and then washed with PBS. 0.2% of Triton X‐100 was permeabilized at room temperature for 45 min, 2% of BSA‐TBSTT solution was incubated at room temperature for 45 min, and then washing with TBST three times. Cyt c, 8‐OHdG, Insulin, and Glucagon primary antibody dilutions were used to cover the surface of the sections and incubated overnight at 4 °C. The sections were equilibrated to room temperature and incubated with Alexa Fluor‐488‐coupled goat anti‐rabbit antibody wet box at 37 °C for 1 h. The nuclei were stained with DAPI dropwise and incubated for 3 min at room temperature, and sealed with the anti‐fluorescence bursting agent ProLong Glass Antifade Mountant (invitrogen). The tissue sections were then imaged using a Leica Confocal Laser Scanning Microscope (CLSM) SP8 inverted confocal microscope under three separate laser lines (552, 405, and 488 nm) of excitation.