Rabbit anti mnsod 1 100

Cells were plated and grown on confocal dishes (MatTek, Ashland, MA) in standard media to appropriate (60–80%) confluence. Cells were then washed three times for 3 min with 1% PBS and fixated in 4% paraformaldehyde (Sigma) for 10 min. Cells were washed three times in 1% PBS for 3 min and permeabilized in 100% methanol for 15 min. Cells were washed in 1% PBS three times for 3 min each and then blocked using 5% bovine serum albumin (BSA, Sigma) for 60 min. Cells were then washed in 1% TBS-T three times for 3 min and incubated in primary antibodies [rabbit anti-Cav-1–1:100 (Abcam), mouse anti-Cav-1–1:100 (BD Technologies), rabbit anti-MnSOD- 1:100 (Abcam), goat anti-MnSOD-1:100 (Santa Cruz), rabbit anti-Nrf2–1:100 (Santa Cruz)] in 1% BSA in 1% TBS-T overnight at 4°C in a humid chamber. Cells were washed three times in 1% TBS-T for 3 min and incubated with secondary fluorescent antibodies [rabbit Alexfluor-568, mouse Alexafluor-488 and goat Alexafluor-647 (Thermo Fisher Scientific)] for 2 h at room temperature in a dark, humid chamber. Cells were then washed three times in 1% TBS-T for 3 min and incubated with 50 μM DAPI (Thermo Fisher Scientific) for 30 min. Cells were washed twice in 1% TBS-T for 3 min and once in PBS for 5 min and then imaged using LSM-510META (Carl Zeiss Microscopy, Thornwood, NY).

Antigen retrieval was done using 10 mM Sodium Citrate buffer at 20 psi for 5 min in a Decloaking Chamber electric pressure cooker (Biocare Medical, Walnut Creek, CA). Slides were blocked with 10% FBS for 45 min and incubated with primary antibody [rabbit anti-Cav-1–1:100 (Abcam), mouse anti-Cav-1–1:100 (BD Technologies), rabbit anti-MnSOD-1:100 (Abcam), goat anti-MnSOD-1:100 (Santa Cruz), rabbit anti-Nrf2–1:100 (Santa Cruz)] overnight at 4°C in a humid chamber. Non-immune IgG was used for negative control. After rinsing in TBS-T, sections were incubated with secondary antibody [rabbit Alexfluor-568 and mouse Alexafluor-488 (Thermo Fisher Scientific)] for 2 h at room temperature in a dark, humid chamber. After incubating with 50 μM DAPI, slides were mounted with FluoroMount Aqueous Solution (Sigma) and examined on an Apotome fluorescen microscope (Zeiss).