4 6 diamidino 2 phenylindole dihydrochloride stock 14.3 mm dapi

Cells grown on coverslips were rinsed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in PBS for 15 min at 25°C, and then permeabilized by 5 min of treatment in −20°C methanol. After incubation in blocking buffer (20% normal goat serum in PBS) for 1 h at 25°C, the cells were exposed to α-Matrin primary antibody at a 1:200 dilution (ab 151714, Abcam, Cambridge, MA) overnight at 4°C. After three 5-min washes in PBS, the cells were then incubated for 1h with secondary antibody, Alexafluor goat anti-rabbit 568, before three washes in PBS (5-min each). A final incubation with 4’,6-diamidino-2-phenylindole, dihydrochloride, stock 14.3 mM (DAPI) (Invitrogen, Carlsbad, CA) for 10’ was followed by one 5-min wash in PBS. Cells were then mounted with Vectashield (Vectorlabs, Burlingame, CA, USA) mounting media for fluorescence and viewed using an Olympus BX60 microscope. Images were obtained using a digital camera Olympus DP71 and Micro DP-BSW controller software. The analysis of Matrin 3 localization was performed by an investigator that was blind to cell genotype.

After rinsing the cells 3 times in 1× PBS, transfected cells were fixed with 4% paraformaldehyde in 1× PBS for 15 minutes. Cells were then permeabilized using ice-cold 100% methanol for 5 minutes followed by incubation in 20% normal goat serum in 1× PBS. Immunostaining was then performed with hSOD1 (1:1000) and C4F6 (1:1000) antibodies in 1× PBS with 10% normal goat serum and incubation overnight at 4°C. Cells were then incubated for 1 hour with secondary antibodies (Invitrogen, Carlsbad, CA) Alexafluor goat anti-rabbit 568 for hSOD and Alexafluor goat anti-mouse 568 for C4F6 diluted 1:2000 in 1× PBS with 10% normal goat serum. Cells were treated with 4’,6-diamidino-2-phenylindole, dihydrochloride, stock 14.3 mM (DAPI) (Invitrogen, Carlsbad, CA) diluted 1:2000 in 1× PBS for 10 minutes to visualize nuclei. Fluorescence was visualized on an epifluorescence Olympus BS60 microscope.

Cells were exposed to sodium (meta) arsenite ≥90% (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 100 μM diluted in cell culture media. At 24 h post-transfection, cells were washed in PBS and the media was replaced with media containing sodium arsenite for 45 minutes at 37°C to activate a stress response. The cells were then washed twice with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in PBS for 15 min at 25°C, rinsed three times with PBS (10 minutes each wash), treated with 4’,6-diamidino-2-phenylindole dihydrochloride stock 14.3 mM (DAPI) (Invitrogen) for 10 minutes, followed by a final 5-min wash in PBS. Cells, fixed to the coverslips, were then mounted with Vectashield (Vectorlabs) mounting media for fluorescence and were imaged 24–36 h after transfection for co-localization using an Olympus BX60 microscope. Images were obtained using a digital camera Olympus DP71 and Micro DP-BSW controller software.