Western blot analysis of ADA in Lane 1: mouse testis tissue lysate, Lane 2: mouse ovary tissue lysate, Lane 3: rat stomach tissue lysate, Lane 4: rat ovary tissue lysate using 50 µg (reducing conditions) per well. Electrophoresis was performed on 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours and protein was transferred to a nitrocellulose membrane at 150mA for 50-90 minutes. Sample was blocked with 5% Non-fat Milk/TBS for 1.5 hours at room temperature, incubated with ADA polyclonal antibody (Product # PA5-78722) at a dilution of 0.5 µg/mL (overnight at 4°C), followed by goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10,000. Signal development was performed using a chemiluminescence (ECL) kit.

Immunohistochemistry analysis of ADA on paraffin-embedded mouse intestine tissue. Antigen retrieval was performed using citrate buffer (pH6, epitope retrieval solution) for 20 mins. Sample was blocked using 10% goat serum, incubated with ADA polyclonal antibody (Product# PA5-78722) with a dilution of 1 µg/mL (overnight at 4°C), and followed by biotinylated goat anti-rabbit IgG (30 minutes at 37°C). Development was performed using Streptavidin-Biotin-Complex (SABC) with DAB chromogen method.