Ab84952

Slides from TMAs (4 μm-thick) were used for immunohistochemistry (IHC) reactions, which were performed using DakoAutostainer Link48 (Dako, Glostrup, Denmark). In order to deparaffinize, rehydrate and unmask the antigens the sections were boiled in EnVision FLEX Target Retrieval Solution (pH 9, 20 min, 97 °C; Dako) using the PTLink platform (Dako, Glostrup, Denmark). Afterwards, slides were incubated for 5 min with Envision Flex Peroxidase-Blocking Reagent (Dako, Glostrup, Denmark) to block endogenous peroxidase. As primary antibodies (20 min, RT), rabbit polyclonal antibodies against AMH (1:100, ab84952, Abcam, Cambridge, UK) were used. Next, slides were incubated with EnVision FLEX/HRP (20 min, RT), and the reaction was visualized (10 min, RT) with freshly prepared 3,3′-diaminobenzidine (DAB). Additionally, slides were counterstained for 5 min with EnVision FLEX Hematoxylin (Dako, Glostrup, Denmark). Finally, slides were dehydrated in ethanol (70%, 96%, absolute) and xylene, then mounted with Dako Mounting Medium (Dako, Glostrup, Denmark). Slides were evaluated using the Olympus BX41 light microscope (Olympus, Japan). Control tissues included the human prostate.

ELISA was used to determine the EC₅₀ of the B10 antibody. A 96-well high protein-binding capacity plate (Nunc MaxiSorp) was coated with polyclonal anti-AMH antibodies (Abcam ab84952) overnight. Then, the plate was washed 3 times and saturated with a PBS/0.01% Tween-20/2% BSA solution for 2 h. After each step, the plate was washed 3 times with PBS/0.01% Tween-20. After incubation with LR-AMH (25 nM) at 37 °C for 2 h, the B10 antibody (666–0 nM) was added at 37 °C for 1h30 followed by the secondary anti-Fc mouse peroxidase (HRP) antibody for 30 min, and finally the substrate enzyme (Thermofisher TMB). Absorbance was read at 450 nm after stopping the enzymatic reaction by the addition of sulfuric acid.