K4006

In all, 5 μm sections were cut using a microtome. Sections were floated onto slides and left to air-dry. Sections were then baked overnight at 65 °C. Samples were deparaffinised by passing through xylene and 100 and 70% ethanol washes. All staining was performed using the DAKO EnVision+ System-HRP (DAB) kit for rabbit (K4010; DAKO) or mouse (K4006; DAKO) primary antibodies. The following primary antibodies were used: ProCaspase-3 (1 : 100; Rabbit polyclonal, HPA002643, Atlas Antibodies), Active Caspase-3 (1 : 200; Rabbit polyclonal, 9661, CST), COX-2 (1 : 400; Mouse monoclonal; CAY160112-1, Cayman (@Cambridge Bioscience)), β-Catenin (1 : 300; Neomarkers RB-9035 (Fisher Scientific, Fremont, CA, USA)), Ki-67 (MIB1) (1 : 100; Dako, M7240), ZEB1 (1 : 100; Rabbit polyclonal, HPA027524, Atlas Antibodies). Positive and negative controls were included according to the suppliers′ recommendations (Negative Control Rabbit Immunoglobulin Fraction (Normal), X 0903, Dako; Negative Control Mouse IgG1, X0931, Dako). Following staining, sections were scored semi-quantitatively, on a scale of 0–3 based on the extent and intensity of staining, as described above.

For a pilot study, formalin-fixed paraffin-embedded human lung tissues obtained from two SSc-PAH patients were used for immunohistochemistry studies. Briefly, the lung tissues were cut at 3 microns sections, mounted on glass slides, and deparaffinized. For RAGE immunohistochemistry, antigen retrieval was performed with tris-HCl and EDTA. Mouse-anti-human RAGE (Santa Cruz, (A-9): sc-365154, Dallas, Texas, USA) was added overnight. Consecutively, samples were incubated with rabbit anti-mouse-HRP conjugate (Dako, 0260, Santa Clara, USA), and stained with diaminobenzidine as a substrate (Dako K4006, Santa Clara, USA). Negative control staining was performed by omission of RAGE antibody showing no aspecific binding. For determining RAGE localization, additional staining was performed on serial sections as follows. For comparison, standard histochemical Haematoxylin-Eosin and Verhoeff’s elastin stains were performed, in addition to, immunohistochemical stains for CD31 (endothelial cells), alpha-smooth muscle actin ([α-SMA] myofibroblasts and smooth muscle cells), and desmin (smooth muscle cells). The latter immunohistochemical stains were performed using an automated Ventana Benchmark Ultra immunostainer (Roche Diagnostics Netherlands) in our ISO15189 accredited pathology department.