One aortic valve from an AS patient was processed with this methodology. After surgical sample collection, valve leaflets were fixed in 10% buffered formalin for 24 h and processed for paraffin embedding using standard procedures for histological and IHC analysis. The specimens were sectioned at 4 µm thickness and stained with haematoxylin–eosin (H&E). Decalcification with a 10% EDTA solution was done if needed.
For IHC analysis, 3 µm-thick sections were obtained, and immunostaining was performed using the rabbit monoclonal antibody anti-CD3 (1:150) (Abcam ab16669, Cambridge, UK) with the Envision FLEX/HRP system (Dako, Glostrup, Denmark). For IHC staining, the secondary antibody (Envision FLEX/HRP) was used for 30 min at room temperature, followed by 3,3′-diaminobenzidine (DAB) staining (Dako, Glostrup, Denmark) before being counterstained with Harris haematoxylin. Human lymph node was used as a positive control for CD3 antibody and staining in the absence of the primary antibody was used as a negative control. IHC images were acquired using a Leica microscope (Leica Microsystems, Wetzlar, Germany) and a Thunder Imager microscope (Leica Microsystems, Wetzlar, Germany), respectively.
For IHC analysis, 3 µm-thick sections were obtained, and immunostaining was performed using the rabbit monoclonal antibody anti-CD3 (1:150) (Abcam ab16669, Cambridge, UK) with the Envision FLEX/HRP system (Dako, Glostrup, Denmark). For IHC staining, the secondary antibody (Envision FLEX/HRP) was used for 30 min at room temperature, followed by 3,3′-diaminobenzidine (DAB) staining (Dako, Glostrup, Denmark) before being counterstained with Harris haematoxylin. Human lymph node was used as a positive control for CD3 antibody and staining in the absence of the primary antibody was used as a negative control. IHC images were acquired using a Leica microscope (Leica Microsystems, Wetzlar, Germany) and a Thunder Imager microscope (Leica Microsystems, Wetzlar, Germany), respectively.