Fluoromont g mounting medium

Cells were treated with DMSO (1% v/v), TGFβ1 (5 ng·mL⁻¹), and/or NOX1/4 inhibitor (GKT137831) (20 µm) during 24h in N2B27 medium. After treatment, cells were washed with PBS and fixed with 3.7% (w/v) paraformaldehyde in PBS for 15 min. Subsequently, cells were washed with PBS and blocked with 10% FBS in PBS with 1% BSA for 1 h, at RT. Then, cells were permeabilised with 0.1% Triton‐X‐100 in 0.1% BSA for 5 min. Next, cells were incubated at RT for 2 h with primary antibodies against Ki67 (1 : 1000 in PBS containing 1% BSA, rabbit, ab15580; Abcam^®). Afterwards, cells were washed in PBS and incubated for 1 h in dark at RT with donkey anti‐Rabbit IgG Alexa Fluor 488 secondary antibody (1 : 200 in PBS with 1% BSA, A21206; Thermo Fisher Scientific). Then, cells were incubated in dark at RT for 5 min with DAPI (1 : 1000 in PBS with 1% BSA; Sigma‐Aldrich Co.). Later, cells were washed three times with PBS and the coverslips were mounted on the slides using Fluoromont‐G^® mounting medium (Southern Biotech, Birmingham, AL, USA) and dried in dark at RT. Pictures were acquired with the fluorescence microscope Eclipse 90i and processed using NIS‐Elements software (Nikon, Tokyo, Japan). The quantification of the proliferative phenotype of cells was performed by quantifying Ki67‐positive cells, and total number of nuclei per picture using fiji image j software [35 (link)].

For the microscopic analysis of autofluorescence, cells were grown on coverslips for 16hr, treated with 50 μM for 3 h before being fixed with 4% PFA. After mounting with Fluoromont-G mounting medium (Southern biotech), coverslips were observed with a Zeiss confocal microscope and fluorescence spectra induced by menadione treatment was evaluated in multiple regions of interest (ROI) within the same cell.
For indirect immunofluorescence analysis, cells were fixed on coverslips for 15 min with 4% PFA and then permeabilized for 5 min with Triton 0.2% at room temperature. AIF protein was detected by incubation with an anti-AIF pAB (1:100 dilution in PBS / 1% BSA / 2% goat serum) followed by Alexafluor-647-conjugated goat anti-rabbit (Life Technologies). DNA was stained with Hoechst 3342 or TO-PRO-3 (Life Technologies). Sample slides were then mounted using the reagent Fluoromont-G (Southern Biotech) and observed by confocal microscopy (LSM-510; Carl Zeiss Microimaging) equipped with an X63 objective.