Four-micrometer-thick sections of f

Four-micrometer-thick sections of formalin-fixed, paraffin-embedded tissue of patients with OSCC on Superfrost Plus slides were subjected to immunohistochemical staining. From patients where several tumour-containing paraffin-blocks were available, a block with representative material, based on H/E staining, was chosen without specific evaluation of the inflammatory infiltrate. Before staining, all specimens were incubated overnight at 60°C, deparaffinised in xylene, rehydrated in graded alcohol baths and subjected to heat-induced antigen retrieval in 0.01 M sodium citrate buffer at pH 6.0. Prior to antibody incubation, inherent peroxidase activity in the tissue was blocked with 3% H2O2 (Ventana Medical Systems, France or Dako Glostrup, Denmark). The following primary antibodies were used: Mouse anti-CD20, clone L26; Mouse anti-CD21, clone 2G9; Mouse anti-bcl-6, clone GI191E/A8; Mouse anit-CD34, clone QBEnd/10; Rabbit anti-CD3, clone 2GV6 (all from Ventana Medical Systems, France); Mouse anti-Podoplanin, clone D2-40 (Dako, Glostrup, Denmark) and Rat anti-PNAd, clone MECA-79, (Biolegend, San Diego). Dilutions and incubation times are listed in Table 2. Except for the PNAd antibody, all immunohistochemical stainings were done in the automated slide stainer Ventana Benchmark, XT (Ventana) at the Diagnostic Clinic – Clinical Pathology, UNN, which is accredited according to the ISO/IEC 15189 standard for the respective stainings, using the same protocols, positive and negative controls as in the clinical routines. For these automated stainings, a cocktail of HRP labelled goat anti-mouse IgG/IgM and mouse anti-rabbit secondary antibodies together with diaminobenzidine from the Ventana UltraView Universal DAB Detection Kit (#760-500, Ventana) were applied for visualization.