Mouse anti tom20 antibody

HEK293 cells cultured on coverslips were transiently transfected with WT or K4R SHLP2-EGFP for 36 hr and were cultured on coverslips and then fixed with 4% paraformaldehyde for 10 min at room temperature. After fixation, the cells were permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 10 minutes at room temperature and were blocked in PBS containing 0.2% Triton X-100 and 1% bovine serum albumin (BSA) for 1 hour at room temperature. Cells were then incubated with mouse anti-Tom20 antibody (1:100; Santa Cruz Biotechnology) in PBS containing 0.2% Triton X-100 and 1% BSA at 4 °C overnight. After three washes with PBS, the cells were further incubated with Alexa Fluor 568-conjugated donkey anti-mouse IgG (1:200; Invitrogen) in PBS containing 0.2% Triton X-100 and 1% BSA for 1 hour at room temperature in dark. Nuclei were stained for 5 minutes at room temperature in PBS containing Hoechst 33258 (2 mg/ml; Invitrogen). Coverslips were mounted with ProLong Gold antifade reagent (Invitrogen). Images were acquired with a Keyence microscope (Keyence corporation of America, Itasca, IL). For immunostaining of COX IV antibody, fixed and permeabilized cells were incubated with anti-COX IV antibody (1:200; abcam) and Alexa Fluor 448-conjugated donkey anti-rabbit IgG (1:200; Invitrogen).

Primary dermal fibroblast cells on coverslips were fixed with 4% paraformaldehyde for 10 min at room temperature. After fixation, the cells were permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 10 minutes at room temperature and were blocked in PBS containing 0.2% Triton X-100 and 1% bovine serum albumin (BSA) for 1 hour at room temperature. Cells were then incubated with rabbit anti-GRSF1 antibody (1:100; Sigma) or mouse anti-Tom20 antibody (1:100; Santa Cruz Biotechnology) in PBS containing 0.2% Triton X-100 and 1% BSA at 4 °C overnight. After three washes with PBS, the cells were further incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:200; Invitrogen) or Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:200; Invitrogen) in PBS containing 0.2% Triton X-100 and 1% BSA for 1 hour at room temperature in dark. Nuclei were stained for 5 minutes at room temperature in PBS containing Hoechst 33258 (2 mg/ml; Invitrogen). Coverslips were mounted with ProLong Gold antifade reagent (Invitrogen) and observed under an LSM780 confocal microscope (Carl Zeiss, Germany).

Primary dermal fibroblasts plated on coverslips were fixed with 4% paraformaldehyde for 10 min at RT. After fixation, cells were permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 10 min at RT and blocked in PBS containing 0.2% Triton X-100 and 1% bovine serum albumin (BSA) for 1-h at RT. Cells were then incubated with mouse anti-Tom20 antibody (1:100; Santa Cruz Biotechnology) in PBS containing 0.2% Triton X-100 and 1% BSA at 4° C overnight. After three washes with PBS, cells were incubated with Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:200; Invitrogen) and Alexa Fluor 568-conjugated donkey anti-rabbit IgG (1:200; Invitrogen) in PBS containing 0.2% Triton X-100 and 1% BSA for 1-h at RT in the dark. Nuclei were stained for 5 min at RT in PBS containing Hoechst 33258 (2mg/ml; Invitrogen). Coverslips were mounted with ProLong Gold antifade reagent (Invitrogen) and observed under an LSM780 confocal microscope (Carl Zeiss, Germany).