Cellsans software

Paraffin-embedded sections underwent deparafinization by Histoclear and a graded series of ethanol washes (100%, 95%, 75% and 50%), followed by antigen retrieval in Antigen Unmasking Solution, Citric Acid Based (Vector Laboratories, Burlingame, CA), in a steamer for 20 minutes, followed by blocking of non-specific binding using 10% normal goat serum (NGS). Slides were incubated with rabbit-anti-ki67 (1:100) (Thermo Fisher Scientific, Waltham MA) primary antibody at 4°C overnight. Slides were washed 3 times with 0.1 PBS + 0.01% Tween-20 buffer, then incubated with fluorochrome conjugated Alexa Fluor 555 Goat Anti-Rabbit secondary antibody (1:400) (Life Technologies, Grand Island, NY) at room temperature for 1 hour. Slides were mounted with cover slips using ProLong Gold Antifade Mounting medium with Dapi (Life Technologies, Grand Island, NY) mounting solution. Slides were examined for Ki67 staining using the Leica DMRX microscope equipped with fluorescence capabilities. Images were acquired with the DP72 camera (Olympus) using the CellSans software (Olympus).

Adrenal glands that were dissected from mice were fixed in 10% neutral buffered formalin, embedded into paraffin blocks and sectioned (5 μm) and onto glass slides, following standard procedures by Histoserv Inc (Germantown, MD). Histological staining using haematoxylin/eosin (H&E) staining was also performed (Histoserv Inc., Germantown, MD). Immunohistochemistry (IHC) was performed in house for Caspase-3 (rabbit-anti-cleaved-caspase 3 antibody, 1:400, Cell Signaling Technology, Danvers, MA) using the Rabbit ImmPRESS Kit (Vector Laboratories, Burlingame, CA), following manufacturers protocol. Staining was visualized using ImmPACT DAB Peroxidase (HRP) Substrate (Vector Laboratories, Burlingame, CA). Coverslips were mounted onto the stained sections using VectaMount AQ Aqueous Mounting Media (Vector Laboratories, Burlingame, CA). Slides were examined using the Leica DMRX microscope. Images were acquired with the DP72 camera (Olympus) using the CellSans software (Olympus).