Myocilin sc 515500

Primary HTM cells were plated in a confocal dish at a density of 8.0 × 10⁴ per dish, infected with lentivirus carrying empty vector, Myoc-WT, or Myoc-N450Y at an MOI of 30, and cultured for 72 h. Then, the supernatant was discarded, and the cells were washed with PBS (1X; 155 mM NaCl, 3 mM Na₂HPO₄, 1 mM KH₂PO₄, pH 7.4). The cells were fixed with 4% paraformaldehyde (CWBIO) for 20 min, permeabilized with 0.3% TritonX-100 (CWBIO), and blocked with 5% bovine serum albumin (BSA) for 1 h. Primary antibody myocilin (sc-515500, Santa Cruz Biotechnology) and calreticulin (12,238, CST) were diluted in 0.5% BSA at a concentration of 1:100. Then, the cells were incubated with primary antibodies at 4 °C overnight, followed by a 1-h incubation with Alexa Fluor 488 (A21206; Thermo) and Alexa Fluor 546 (A10036; Thermo) secondary antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Peterborough, UK). Images were captured with a Zeiss and Leica imaging system (Carl Zeiss; Hertfordshire, UK).