Fluoroshield mounting media solution

BG4 immunofluorescence was performed as previously described^{79 (link)}. Briefly, cells were seeded in 6- or 24-well plates. 24 h post seeding the cells were treated with 2 µM PDS for 48 h and pre-fixed with a 50/50 solution of DMEM and methanol/acetic acid (3:1) at RT for 5 min. After a brief wash with methanol/acetic acid (3:1) the cells were fixed with methanol/acetic acid (3:1) at RT for 10 min. Cells were then permeabilized with 0.1% (v/v) Triton X-100 in PBS at RT for 3 min with gentle rocking and incubated with a blocking solution (2% (w/v) dry milk in PBS, pH 7.4) for 1 h at RT with gentle rocking. Afterwards cells were incubated in blocking solution containing 0.5–1 μg of BG4 antibody per slide and incubated for 2 h at RT. Cells were then incubated with blocking solution containing 1:800 rabbit polyclonal antibody against the DYKDDDDK epitope (Cell Signaling #2368) for 1 h at RT with gentle rocking. Next, cells were incubated at RT with blocking solution containing 1:1000 fluorescent secondary anti-rabbit IgG (Life technologies #A10520) for 1 h at RT with gentle rocking. After each step, cells were washed three times with 0.1%(v/v) Tween-20 in PBS for 10 min. The cover glasses were mounted with a drop of Fluoroshield mounting media solution (Merck) containing the DNA staining fluorophore DAPI.

BG4 immunofluorescence was performed as previously described [13 (link)]. Briefly, cells were seeded in 6- or 24-multiwell plates; 24 h post seeding cells were treated with PDS and CX-5461 and pre-fixed with a 50/50 solution of DMEM and methanol/acetic acid (3:1) at RT for 5 min. After a brief wash with methanol/acetic acid (3:1), the cells were fixed with methanol/acetic acid (3:1) at RT for 10 min. Cells were then permeabilized with 0.1% (v/v) Triton X-100 in PBS at RT for 3 min under gentle rocking and incubated with blocking solution (2% (w/v) dry milk in PBS, pH 7.4) for 1 h at RT under gentle rocking. Afterwards, cells were incubated in blocking solution containing 0.5/1 μg of BG4 antibody per slide and kept 2 h at RT. Cells were then incubated with blocking solution containing 1:800 rabbit polyclonal antibody against the DYKDDDDK epitope (Cell Signalling ref #2368) for 1 h at RT under gentle rocking. Next, cells were incubated at RT with blocking solution containing 1:1000 fluorescent secondary anti-rabbit IgG (Life technologies ref #A10520) for 1 h at RT under gentle rocking. After each step, cells were washed three times with 0.1% (v/v) Tween-20 in PBS for 10 min. The cover glasses were mounted with a drop of Fluoroshield mounting media solution (Merck) containing the DNA staining fluorophore DAPI.