Immunofluorescent Localization of Adrenal Enzymes

As described previously (23 (link)), adrenal glands were sliced into 6-µm thick sections using the cryostat microtome (Thermo Scientific) and sections were place in ice-cold phosphate-buffered saline (PBS). Tissue sections were permeabilized with PBS including 0.1% Tween detergent for 5 minutes. Tissues were blocked with 5% goat serum and then incubated with the 1:100 diluted CYP11B1 (RRID: AB_3076259) and CYP11B2 (RRID: AB_3076258) antibodies (Gomez-Sanchez) in 1% bovine serum albumin (w/v) in PBS-T overnight at 4 °C. Sections were washed with PBS 3 times and incubated with 1:500 diluted antimouse (RRID: AB_2338902)and antirabbit (RRID: AB_2338078) Alexa 647 (Jackson), respectively. For nuclear counterstaining, 20 µM Hoechst 33342 (Invitrogen H3570) was used. After washing 3 times, coverslips were mounted onto the slides with mounting medium FluorSave (Millipore 345 789).