2.2. Histopathology and Immunohistochemistry

The formalin-fixed, paraffin-embedded blocks of 60 tissue samples collected from patients of the study group were sectioned (2 µm). Normal oral mucosa (NOM) in margins of 7 formalin-fixed, paraffin-embedded archival blocks of fibromas were used as controls. For histopathological examination, the sections were stained with hematoxylin and eosin (H&E). OSCCs were graded as well, moderately, and poorly differentiated using the standard WHO criteria [39]. The criterion for judging the malignant potential of OPMDs is mainly the presence and degree of dysplasia [39]. OPMDs were classified histologically into stages with increasing risk of developing into OSCC, namely as mild, moderate, and severe epithelial dysplasia according to the WHO criteria [6,40].

For immunohistochemistry, the sections were deparaffinized and rehydrated. After antigen retrieval, slides were incubated with antibodies: Anti-IL-1α rabbit polyclonal IgG (30 min., room temp.) and Anti-IL-6 mouse monoclonal IgG (30 min., room temp.) purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA) and Anti-IL-8 mouse monoclonal IgG (60 min., room temp.) and Anti-TNF-α rabbit polyclonal IgG (60 min., room temp.) purchased from Abcam Plc. (Cambridge, UK) diluted (1:50) in Tris-buffer solution (TBS) followed by incubation with Large Volume HRP Polymer (Lab Vision Corporation, Fremont, CA, USA). For visualization of the antigen 3,3-diaminobenzidine tetrahydrochloride (Lab Vision Corporation, Fremont, CA, USA) was added. Slides were counterstained with hematoxylin (Thermo Fisher Scientific, Waltham, MA, USA).

Taking into account that epithelium and stroma contain completely different cells, we analyzed them separately. Depend on the proportion of the positively stained cells and intensity of staining, a semiquantitative immunoreactive score from 0 to 6 was calculated separately for epithelial/cancer cells and stromal cells (Table 1). The overall score was not calculated. The score was elaborated by authors based on the literature [41,42,43].

Immunoreactive score for cytokines immunohistochemistry.

All histopathological and immunohistochemical evaluations were made by board-certified specialist in pathomorphology (K.O.) in the Chair of Pathomorphology, Jagiellonian University Medical College, Krakow, Poland.