Mouse anti vimentin v9 mab

MDA-MB-231 cells were seeded on 13-mm glass coverslips in a 24-well culture plate in the presence or absence of BZ, PTX, and VNR and cultured for 24 h. Coverslips were washed twice with PBS and fixed for 15 min on ice in methanol. After washing twice with PBS, cells were permeabilized with 0.1% Triton X-100 in TBST for 5 min at room temperature. The coverslips were then washed with TBST and further incubated with TBST containing 10% normal goat serum (NGS; Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature for blocking nonspecific binding. Cells were incubated with a primary antibody such as mouse anti-vimentin (V9) mAb, mouse anti-ubiquitin (P4D1) mAb, and anti-p62/SQSTM1 (D-3) mAb (all from Santa Cruz), and diluted in TBST, 1.5% NGS, 0.1% bovine serum albumin (BSA) at 4°C overnight. The coverslips were washed three times for 5 min with TBST at room temperature and subsequently incubated with Alexa Fluor^® 488 conjugate-goat anti-mouse IgG (H+L) secondary antibody (Thermo Fisher Scientific), diluted in TBST containing 1.5% NGS and 0.1% BSA, for 45 min at 37°C. The coverslips were washed with TBST and mounted in ProLong^® Diamond Antifade Montant (Thermo Fisher Scientific). Nuclei were stained with DAPI (Sigma-Aldrich, D-9542), and the cells were imaged using a confocal laser scanning fluorescence microscope, LSM 700 (Carl Zeiss, Germany).