Fv10i liv confocal microscope

CGN were plated at a density of 50 × 10³ cells/cm² and were grown onto poly-l-lysine-coated glass slides from 0 to 3 DIV. Cells were washed twice in PBS and fixed with 4% paraformaldehyde for 20 min. Subsequently, cells were blocked and permeabilized overnight at 4℃ in blocking solution (PBS containing 0.5% Triton X-100 and 10% normal goat serum) and then were incubated overnight at 4℃ in blocking solution with 1:250 (0.4 µg/ml) rabbit anti-NOX2 and 1:500 mouse anti-Tau antibodies. The primary antibody anti-NOX2 was detected with an Alexa Fluor® 488 goat anti-Rabbit IgG (H+L) secondary antibody (1:1000) incubated for 2 hr at room temperature. The primary antibody anti-Tau was detected with a DyLight 594 goat anti-Mouse IgG (H+L) secondary antibody (1:1000) incubated for 2 hr at room temperature. Coverslips were mounted using Vectashield mounting media with DAPI. Images were captured in the FV10i-LIV confocal microscope (Olympus, USA). Coverslips incubated without primary antibody or with 1:50 (10 µg/ml) normal rabbit IgG showed no staining. The staining for Tau detection did not affect the detection of NOX2 and vice versa.

MCF-7 cells grown on coverslip were incubated with STS (24 µM) for 48 h. Cells were fixed with 4% paraformaldehyde (FUJIFILM Wako Pure Chemical Corporation) and permeabilized with 0.2% Triton X-100 (FUJIFILM Wako Pure Chemical Corporation). After blocking with 3% bovine serum albumin (BSA: Sigma-Aldrich) in PBS, cells were incubated for 1 h with primary antibodies against Proliferating Cell Nuclear Antigen (PCNA) and cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA) and wash with 0.1% Tween 20 in PBS. Then, cells were treated with Alexa Fluor 488- and 568-conjugated goat anti-mouse and rabbit secondary antibody (Thermo Fisher Scientific). The nuclei were stained with DAPI (FUJIFILM Wako Pure Chemical Corporation). After washing, laser scanning confocal microscopy was performed using FV10i-LIV confocal microscope (Olympus Corporation, Tokyo, Japan). To quantify PCNA expression, fluorescence intensity of nuclear PCNA was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Staurosporine (FUJIFILM Wako Pure Chemical Corporation) was used as the positive control for apoptosis.

Monolayer cultures of cells were stained with 1 μM solutions of CellTracker Green CMFDA or Red CMTPX (Invitrogen) for 30 min in a CO₂ incubator. Equal volumes of single-cell suspensions of red and green-labeled cells at densities of 1.0 × 10⁵ cells/ml were mixed in growth media containing 0.5% methylcellulose, and placed on polyHEMA-coated plates. Aliquots of suspended cells were withdrawn at regular intervals and stained with Hoechst 33342 (10 μM; Sigma-Aldrich) for 15 min at room temperature, washed with PBS, and fixed with 3.7% formaldehyde for 10 min at room temperature. Fixed cells were washed with PBS and placed in glass bottom dishes.
Laser scanning confocal microscopy was performed using FV10i-LIV confocal microscope (Olympus Corporation, Shinjuku, Tokyo, Japan). Three-dimensional images were acquired through z-stacking of sequential optical x-y sections taken at 0.5–1.0 μm z-intervals. Orthogonal slice views from z-stack images were processed with the FLUOView software (Olympus).

Mouse intestinal organoids were treated with Cy3-labeled SubAB in IntestiCult^TM Organoid Growth Medium for 30 min on ice, washed with cold PBS and then fixed on coverslips using Smear Gell (GenoStaff Inc. Japan).
Cy3-labeled SubAB treated mouse intestinal organoids were incubated at 37 °C for 2 h on Matrigel matrix-coated coverslips, and then fixed with 4% paraformaldehyde for 1 h. Cells on the coverslips were mounted on glass slides using ProLong Gold antifade reagent with DAPI (Invitrogen). The stained cells were visualized using a FV10i-LIV confocal microscope (Olympus). The images were arranged with Adobe Photoshop Elements 15.