Alexa 555 labeled donkey anti rabbit secondary antibody

Double-immunofluorescence staining was performed using cultured podocytes grown on collagen-coated glass cover slips and frozen mouse kidney sections as described previously [37 (link), 38 (link)]. Briefly, after fixation the cells were incubated with goat anti-nephrin (1:200; R&D, Minneapolis, MN, USA) and rabbit anti-pannexin-1 (1:200) overnight at 4°C. Then, Alexa 488-labeled donkey anti-goat secondary antibody (1:200; Life Technologies, CA, USA) and Alexa 555-labeled donkey anti-rabbit secondary antibody (1:200; Life Technologies, CA, USA) were added to the cell slides and incubated for 1 hour at room temperature. Frozen mouse kidney slides were fixed in acetone, blocked, and then incubated with goat anti-nephrin and rabbit anti-pannexin-1 overnight at 4°C. Then, Alexa 488-labeled donkey anti-goat secondary antibody and Alexa 555-labeled donkey anti-rabbit secondary antibody were added to the cell slides and incubated for 1 hour at room temperature. Slides were then washed, mounted, and observed using a confocal laser scanning microscope (Fluoview FV1000, Olympus, Japan).

Immunofluorescence staining was performed using cultured podocytes grown on collagen-coated glass cover slips and frozen mouse kidney sections as described previously [42 ]. Briefly, after fixation the cells were incubated with rat anti-ZO-1 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA) or rabbit anti-FSP-1 (1:100; Abcam Biotechnology, Cambridge, United Kingdom) overnight at 4 °C. Then, Alexa 488-labeled donkey anti-goat secondary antibody (1:200; Life Technologies, CA, USA) or Alexa 555-labeled donkey anti-rabbit secondary antibody (1:200; Life Technologies, CA, USA) was added to the cell slides and incubated for 1 h at room temperature. Slides were then washed, mounted, and observed using a confocal laser scanning microscope (Fluoview FV1000, Olympus, Japan).