Prolong glass antifade mount with nucblue

WT and LKO cells were cultured in 24-well glass-bottomed plates and treated with a vehicle or APPH. At the end of the treatment, the cells were incubated with EdU (5–ethynyl–2′–deoxyuridine), dissolved in DMSO (final concentration of 10 uM), and fixed with 10% buffered formalin for 15 min. The incorporated EdU was detected using a Click-iT EdU Alex fluor 488 imaging kit according to manufacturers’ instructions (C10632, Thermo fisher, Waltham, MA, USA). Glass wells were covered using a Prolong Glass antifade mount with Nucblue (P36981, Invitrogen, Waltham, MA, USA) and imaged (6 random fields/group from three different coverslips; each field had more than 200 cells) using a fluorescent microscope (Nikon Ti2). The number of cells in each field was counted using ImageJ software. The DAPI (blue) nuclear represented the total number of cells, and Edu (green) nuclear represented proliferating cells. The proliferation rate was calculated as: (EdU⁺ nuclear number/DAPI⁺ nuclear number) × 100%.

6μm-8μm sections of optimal cutting temperature (OCT compound, Fisher Healthcare)-embedded murine spleen and kidneys were cut using a cryostat NX (Thermo Scientific) and immediately fixed in acetone before blocking with 5% FBS/PBS. Primary conjugated antibodies (TCRβ-PE, IgD-FITC, CD1d-AF647, CD35-APC, NP-PE, GL7-FITC, CD138-APC, IgG1-FITC; BD Biosciences and BioLegend) were used to label tissue sections, followed by 5% FBS/PBS wash. Coverslips were mounted using Prolong™ Glass Antifade Mount with NucBlue (Invitrogen) and sections were imaged using a Zeiss fluorescent microscope. Analysis included 3-5 images per section, multiple sections per spleen, multiple animals per organ, as noted in figure legends.