Fp sc5110

The following antibodies and molecules were used (concentrations used are indicated in parentheses): Chicken polyclonal anti-calreticulin (Thermofisher, PA1-902A, 4,4 µg/mL), Rabbit polyclonal anti-ERp57 (Abcam, ab10287, Dilution 1:100), Mouse anti-CD47 conjugated to Alexa Fluor A647 (Santa Cruz, B6H12, 2 µg/mL), Annexin V conjugated to Biotin (Biolegend, 640904, 2.5 µg/mL), Rabbit Polyclonal anti-Chicken IgY labeled with Alexa Fluor 532 (Cohesion Biosciences, CSA3314, 5 µg/mL), Goat polyclonal anti-Chicken labeled with Alexa Fluor 555 (Thermofisher, A21437, 2 µg/mL), Donkey polyclonal anti-Rabbit IgG labeled with FluoProbes 647H (Interchim, FP-SC5110, 10 µg/mL), Streptavidin conjugated to Alexa Fluor 647 (Thermofisher, S32357 - 0,4 µg/mL).

Triple IF was performed using antibodies for CYP11B1 and CYP11B2 and either Dab2, SF-1 or KCNJ5 antibodies. The samples were processed as above, with the exception that the 3 primary antibodies were added concurrently. After washing, a mixture of three secondary antibodies was used, including donkey anti-mouse IgG Alexa Fluor 488, donkey anti-rat IgG Alexa Fluor 594 and donkey anti-rabbit IgG Alexa Fluor 647 (FP-SC5110, Interchim, France) at a dilution of 1:300 for 60 min.
Immunofluorescent images were obtained using a TCS SP8 confocal laser-scanning microscope system (Leica Microsystems). All images were obtained on the Primacen platform.