The tissue was fixed in 10% formalin, processed, embedded in paraffin, sectioned, and stained with hematoxylin-eosin. All of the severe PH cases demonstrated plexiform arteriopathy. Plexiform lesions were defined as intravascular networks of abnormal cells, highlighted by the endothelial cell marker, FVIII-related antigen (FVIII-r.ag). All cases, including the rat lung tissue and the liver hemangioma, were stained for caveolin 1 and 2 (monoclonal antibodies, 1:400 dilution; Transduction Laboratories; Lexington, KY), and five PH blocks were selected for serial sectioning and examination with immunohistochemical staining for endothelial cells, smooth-muscle cells, and caveolin-1.
In the serially sectioned cases, the first slide was immuno-stained for caveolin 1 (clone 2297). The 4-^m paraffin sections were deparaffinized, dried overnight, and microwaved in 0.01 mol/L sodium citrate buffer. After washing in phosphate-buffered saline solution (pH 7.4), conventional immunohistochemical procedure was followed using a commercially available detection system (Vectastain Elite Kit; Vector Laboratories; Burlingame, CA). The peroxidase activity was developed with 3′-3′-diamino-benzidine, and counterstaining with hematoxylin was performed. For control, the primary antibody was replaced by phosphate-buffered saline solution or by nonspecific mouse IgG.
The second slide of each serial sectioned case was stained with FVIII-r.ag (polyclonal antibody, 1:500 dilution; Dako Corporation; Carpinteria, CA), and the third slide of each serial sectioned case was stained with a-smooth-muscle actin (SMA) stain (monoclonal antibody, 1:10000 dilution; Sigma; St. Louis, MO). Heat-induced antigen retrieval using pressure cooker heating in a sodium citrate solution was used to optimize immunostaining. An automatic immunostaining device (Ventana ES; Ventana Medical Systems; Tucson, AZ) was employed for the immunohistochemical staining. Incubations with unrelated antibodies were used as controls for the above methods. Appendix was used as the positive control tissue for a-SMA and FVIII-r.ag. In addition, each lung section provided its own internal control for a-SMA (bronchial smooth-muscle cells) and FVIII-r.ag (alveolar septal capillaries).
Additional slides were stained using HO-1 antibody (SPA-895, rabbit polyclonal, 1:1500 dilution; Stressgen; Victoria, BC, Canada). Heat-induced antigen retrieval in a sodium citrate solution was used. The slides were incubated with the primary antibody overnight at 4°C.