The 5-μm serial coronal sections of the lungs of PAH rats were deparaffinized and washed. Epitope retrieval was performed using Target Retrieval Solution (Dako, Gostrup, Denmark). The sections were then washed with PBS and nonspecific binding was blocked with Protein Block, Serum Free (Dako). Primary antibodies targeting the following proteins were used for immunohistochemistry: α-smooth muscle actin (αSMA, 1:50, mouse, Abcam, Cambridge, UK), proliferating cell nuclear antigen (PCNA, 1:100, mouse, Abcam), HGF (1:50, rabbit, Abcam), vimentin (1:100, rabbit, Abcam), and Texas red (1:200, rabbit, Abcam).
The primary antibody staining was visualized using fluorescence-conjugated secondary antibodies (Life Technologies, Grand Island, NY, USA). Haechst33342 (Dojindo, Kumamoto, Japan) as used after incubation with the secondary antibodies, for nuclear counterstaining21 (link).
The primary antibody staining was visualized using fluorescence-conjugated secondary antibodies (Life Technologies, Grand Island, NY, USA). Haechst33342 (Dojindo, Kumamoto, Japan) as used after incubation with the secondary antibodies, for nuclear counterstaining21 (link).
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