Dako lsab peroxidase kit

In a separate set of immunohistochemistry (IHC) experiments using the Dako LSAB peroxidase kit (Dako, Santa Clara, CA, USA), rat heart sections were incubated for 10 min in −20 °C methanol and 3% hydrogen peroxide. The slides were permeabilized and blocked for 30 min in 1% bovine serum albumin (BSA) (Sigma, Kanagawa Prefecture, Japan) and then incubated for 30 min with primary antibodies against α-SMA (dilution 1:1000, Thermo, Waltham, MA, USA) in antibody diluent (Dako). Slides were rinsed with 1 × PBS and incubated for 30 min with a streptavidin-biotin system (Dako) to detect the signals; brown color development was evaluated following incubation with diaminobenzidine (DAB) substrate-chromogen for 1 min (EnVision/HRP, Dako). Finally, after rinsing with deionized water, the slides were counterstained with hematoxylin, dehydrated, mounted, and coverslipped. Staining of α-SMA was used to indicate RV cardiac fibrosis.

Immunohistochemical analysis of lung tissues was performed with primary antibodies against α-smooth muscle (SM)-actin (Sigma-Aldrich) and Notch3 ICD (Abcam) using the Dako LSAB peroxidase kit (Dako). Staining of α-SM-actin was used to indicate the medial layer of small pulmonary arteries (PAs) for the assessment of medical wall thickness (MWT). For the Notch3 signal, lung tissue sections were incubated with rabbit anti-Notch3 and mouse anti-α-SM-actin antibodies for 1 hour, followed by incubation with Alexa-488-conjugated secondary antibody (green, Invitrogen) for Notch3 or Cy3-conjugated secondary antibody (red, Chemicon) for α-SM-actin at room temperature for 30 minutes, and observed with a Leica TCS SP spectral confocal microscope at Microscope Core Laboratory of Chang Gung Memorial Hospital.