K4010

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.

Immunohistochemical stainings with CD163 for macrophages (1:400 overnight 4° C, monoclonal rabbit anti‐CD163 [C‐terminal], Abcam, ab189915, EPR14643‐36, Cambridge, UK) and myeloperoxidase (MPO) for neutrophils (1:400 overnight 4° C, monoclonal mouse‐anti‐human‐myeloperoxidase antibody, R&D Systems, Inc., MAB3174, 392,105, Minneapolis, USA) as well as a routine toluidine‐blue‐staining for mast cell numbers (0.5% aqueous toluidine blue solution for 24 h) were performed from paraffin‐embedded lesional skin samples (MPO: UV = 36, CSU = 27; CD163: UV = 35, CSU = 27; Toluidine blue: UV = 20, CSU = 18), each section cut at 5 µm. An immunohistochemistry protocol with Polymer‐labelled secondary antibodies (anti‐mouse: EnVision+/HRP mouse, Dako, K400011‐2, Glostrup, Denkmark; anti‐rabbit: EnVision+/HRP rabbit, Dako, K4010, Glostrup, Denmark; both applied for 30 min at room temperature) and AEC + ‐substrate system (Dako, K346111‐2, Glostrup, Denmark, applied for 10 min at room temperature) was used for detecting the primary antibodies. For the immunohistological stainings, photos were taken by an Axioplan‐II‐microscope (Zeiss) at 200× magnification and % of positivity of stained area was assessed by Fiji software.