Immunohistochemical (IHC) staining and IHC evaluation

Formalin-fixed paraffin-embedded 4μm section were immunostained and evaluated as previously described^{[9, 46–49, 54, 55]}. Briefly, heat-induced antigen retrieval was performed using Tris/EDTA buffer and microwave heating for VDR immunostaining, Target Retrieval Solution, pH 9 and PT Link device (Dako, Glostrup, Denmark) for FoxP3 immunostaining and Target Retrieval Solution, pH 9 and PT Link device for other antibodies. The list of primary and secondary antibodies, antigen retrieval protocols and visualization systems are presented in Table 1. Isotopic controls served as negative controls to determine non-specific binding of antibodies. For each immunostaining run also appropriate positive controls were prepared (Table 1).

Characteristic of primary and secondary antibodies and visualization systems used in IHC staining.

ON – overnight;

An observer analyzing immunostained sections was blinded for detailed histopathological diagnosis, malignancy grade/stage and other clinical data, as previously described^{[9, 46–49, 54, 55]}. Briefly, the staining intensity was evaluated with reference to immunostaining of epidermal cells for VDR, RORα, RORγ, CYP27B1, lymph node lymphocytes for FoxP3, kidney cells for CYP24A1, breast cancer cells for HIF-1α, scored as strong. The semiquantitative score, defined by arbitrary units (A.U.) was calculated as: SQ=mean(IR × SI)/100 for VDR, CYP27B1, CYP24A1, HIF1-α, or SQ=mean(IR × SI) for RORα, RORγ and FoxP3, where IR is the percentage of immunoreactive cells and SI is the staining intensity from 0 to 3 arbitrary units (A.U.) with 0 as negative (0), weak (1), moderate (2) and strong (3). The immunostaining cut-off points were as follows: 0–0.99 (no); 1–1.99 (low) and 2–3.0 (high) A.U. for VDR; 0.0–10.0 (no); 10.1–50.0 (low); 50.1–300.0 (high) for RORα; 0–49.99 (no); 50.0–99.99 (low); 100–300 (high) A.U. for RORγ; 0.0–0.99 (no), 1.0–1.99 (low) and 2.0–3.0 (high) A.U. for CYP27B1; 0.0–1.0 (no), 1.1–2.0 (low) and 2.1–3.0 (high) A.U. for CYP24A1; 0–0.99 (no); 1–1.99 (low) and 2–3.0 (high) A.U. for HIF-1α; 0.0–10.0 (no); 10.1–50.0 (low); 50.1–300.0 (high) for FoxP3. Primary and metastatic melanomas were considered as HIF-1α-positive or FoxP3-positive when more than 10% cells showed positive immunostaining.