A1 plus rsi laser scanning confocal microscope

IHC slides underwent deparaffinization and antigen retrieval in Van Andel histology core using the Agilent Target Retrieval Solution, High pH, for Dako PT Link, at 97°C for 20min. These slides were then washed in TBS-T and immersed for 10min at room temperature (RT) in 0.1% Saponin/PBS to permeabilize tissue. After brief washing in TBS-T, tissue was blocked in 5% goat serum/2% BSA/PBS for 1 h at RT in a humidity chamber. After brief washing in TBS-T, slides received primary antibody (Ab) diluted in 1% BSA/PBS and incubated overnight in a humidity chamber at 4°C. Slides were washed with TBS-T the following day and then incubated with secondary Ab and DAPI diluted in 1% BSA/PBS for 1hat RT in the dark. After washing twice with TBS-T and twice with PBS, the tissue was covered with Fluoromount-G mounting medium and a glass coverslip and allowed to solidify for 2h, then sealed with clear nail polish. Once dry, the slides were stored at 4°C in the dark until imaging could be performed using the Nikon A1plus-RSi Laser Scanning Confocal Microscope (Melville, NY) and Nikon Elements acquisition. Laser power, pinhole size, and gain were consistent across samples. All images were deconvolved with Huygens Professional Software (Scientific Volume Image; The Netherlands).