Digest all

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.

Stainings were performed as previously described^{8 (link),9 (link),25 (link)–27 (link)}. Samples were fixed in 4% phosphate-buffered (PBS) paraformaldehyde (pH 7.2—7.4) for 48 h, washed and stepwise dehydrated with an ethanol to xylol gradient. Lung tissue biopsies were then embedded in paraplast and sectioned at 7 μm. Sections were dewaxed, rehydrated and antigens were retrieved by 30 min treatment with pepsin (Digest All; #003,009, Invitrogen) at 37 °C. Sections were washed with water and PBS (pH 7.4), unspecific binding sites were blocked by incubation in PBS supplemented with 0.05% Tween 20 (Sigma) and 5% fetal calf serum for 10 min. Samples were stained with anti-ceramide antibodies (1:100 dilution) or anti-sphingosine antibodies (1:1000 dilution) in H/S (132 mM NaCl, 20 mM HEPES [pH 7.4], 5 mM KCl, 1 mM CaCl₂, 0.7 mM MgCl₂, 0.8 mM MgSO₄) plus 1% FCS at room temperature for 45 min. Sections were washed 3-times with PBS supplemented with 0.05% Tween 20, once with PBS and stained with Cy3-coupled anti-mouse IgM F(ab)₂ fragments diluted 1:200 in H/S, 1% FCS for 30 min. Samples were washed as above and embedded in Mowiol. Sections were analyzed on a Leica TCS-SP5 confocal microscope employing a 40 × lens. Image analysis was performed using a Leica LCS software version 2.61 (Leica Microsystems, Mannheim, Germany) with identical settings for all samples.