C9080

After routine deparaffinization and dehydration, the sections were incubated for 10 min. at 37°C with 1% trypsin (Digest All, cat# 003008; Invitrogen, Darmstadt, Germany) for antigen unmasking. After washing with PBS, the tissues were blocked with 5% BSA for 1 hr at RT and incubated with the appropriate primary antibodies diluted in 3% BSA with 0.2% TritonX-100 in PBS overnight at 4°C. Mouse monoclonal Oct-3/4 (C-10), dilution 1:50 (sc5279; Santa Cruz), mouse monoclonal anti-vimentin, 1:500 (C 9080; Sigma-Aldrich), monoclonal anti-α-actin smooth muscle conjugated with Cy3 (α-SMA), 1:500 (C6198; Sigma-Aldrich), anti-PDGFR-α, 1:100 (ab90967; Abcam, Cambridge, UK), rabbit polyclonal PDGFR-β, 1: 100 (sc-432; Santa Cruz), rat antimouse Ly-6A/E/Sca-1, 1:100 (cat # 553333; BD Biosciences, Heidelberg, Germany), rabbit polyclonal C-kit, 1:100 (sc-168; Santa Cruz), rabbit polyclonal VEGF, 1:100 (sc-152; Santa Cruz) were used for the staining. The sections where then incubated with the appropriate secondary antibodies (1:500–1000) for 1 hr at RT, washed with PBS and counterstained with DAPI (1:1000) for 10 min. The slices were mounted with Mowiol and investigated with a Zeiss LSM 710 laser scanning confocal microscope.

Human donor and IPAH PAAFs were grown to subconfluence in chamber slides, fixed with either acetone-methanol (1:1) or 4% PFA and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich). After blocking (5% BSA in PBS, 1 h), cells were incubated with primary antibodies (HDAC1, HDAC2, HDAC8), followed by Alexa Fluor 488-conjugated secondary antibody (1:1,000) for 1 hr. Negative controls were run in parallel with isotype control IgG (rabbit or mouse or goat) and no primary antibody. The cells were also co-stained for collagen type I (T40777R, Meridian Life sciences) or α-vimentin-Cy3 (1:200, C9080, Sigma). After incubation, slides were counterstained with DAPI (for nuclear staining) and mounted with fluorescent mounting medium (Dako). Images were taken at 400 × magnification with Leica DM6000B (Wetzlar, Germany).