Dylight anti mouse 680

Total protein extracts were prepared from frozen male and female murine kidneys, and subsequently processed for multiplex detection of 11βHSD2 protein together with α-tubulin protein for loading normalization. Immunoblots were incubated overnight in 5% milk/Tris-buffered saline/0.1% Tween 20 with rabbit anti-11βHSD2 (1:1000, Santa Cruz SC-20176, Heidelberg, Germany) and mouse anti-tubulin antibodies (1:5000 Sigma, Saint-Quentin-Fallavier, France) followed by incubation for 1 h at room temperature with secondary antibodies coupled to a fluorochrome, Dylight anti-Rabbit 800 at a dilution of 1:10,000 or Dylight anti-Mouse 680 at a dilution of 1:15,000 (Fischer Scientific, Ilkirch, France). Detection and quantification of specific fluorescent signals was performed in multiplex using an Odyssey Fc (LI-COR, Lincoln, NE, USA).

Total protein extracts were prepared and Western blot analyses were performed as previously described [36 (link)]. Antibodies used were a rabbit polyclonal anti-VDAC antibody (1:500 dilution, Abcam #15895), a rabbit polyclonal anti-TSPO antibody (1:500 dilution, Abcam #109497) and a rabbit polyclonal anti-PISD antibody (1:500 dilution, Abcam #119960) with a mouse monoclonal anti-α-Tubulin antibody (1:10,000 dilution, Sigma-Aldrich) or a mouse monoclonal anti-DRP1 antibody (1:1,000 dilution, Abcam #56788), a mouse monoclonal anti-CYP11A1 antibody (1:500 dilution, Sigma-Aldrich #09166) and a mouse monoclonal anti-ATAD3A antibody (1:1,000 dilution, Abcam #67992) with a rabbit polyclonal anti-GAPDH antibody (1:10,000 dilution, Sigma-Aldrich). Primary incubation was followed by 1 h incubation at room temperature with secondary antibodies coupled to a fluorochrome, Dylight anti-Rabbit 800 at a dilution of 1:10,000 or Dylight anti-Mouse 680 at a dilution of 1:15,000 (Fischer Scientific). Signal fluorescence intensity was detected and measured by Odyssey Fc (Li-Cor Biotechnologies).