Anti acetylated α tubulin antibody

Cells were seeded on coverslips on a 24-well plate at a density of 5 × 10⁴ cells per well and allowed to grow overnight. Next, the medium was replaced with a medium containing the o-AzoCol26DF or the colchicine (without illumination) or DMSO as control and incubated for 24 h under 500 ms pulsed green or blue light irradiation every 15 s. Then, cells were fixed and permeabilised with 100% methanol at −20 °C for 15 min and subsequently washed three times with PBS at room temperature. After 1 h blocking with 3% BSA/PBS at 4 °C, slides were incubated with anti-α tubulin 12G10 antibody (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA) (diluted 1:300 in 3% BSA/PBS) and anti-acetylated α- tubulin antibody (Cell Signalling, Danvers, MA, USA) (diluted 1:1000 in 3% BSA/PBS) overnight at 4 °C. After 3 × 10 min washing with PBS, slides were incubated with AlexaFluor555-conjugated anti-rabbit and AlexaFluor488-conjugated anti-mouse secondary antibodies (diluted 1:400 in 3% BSA/PBS) (Thermo Fisher Scientific, Waltham, MA, USA, A31570) and with DAPI (50 ng/mL) for 1 h at room temperature. After washing (3 × 10 min with PBS), slides were mounted in Fluoromount-G (Southern Biotech., Birmingham, AL, USA). Images were recorded using Leica TCS SP8 (Leica Microsystems, Wetzlar, Germany) confocal microscope and analysed using ImageJ 1.53t software.