Streptabcomplex hrp

Mouse joint samples were fixed, decalcified, embedded with optimum cutting temperature (OCT, Tissue-Tek) and cut into 6 μm sections. Sections were stained with DAPI to detect CFSE⁺ B1a cells in the synovium of knee joint using a confocal microscopy (LSM 710, Zeiss). CXCL13 expression in the synovium of knee joint was detected by immunohistochemical staining. Paraffin-embedded sections of joint tissue were deparaffinized and rehydrated. After endogenous peroxidase inhibition by 0.5% hydrogen peroxide, sections were blocked with rabbit serum and incubated with biotinylated rabbit anti-mouse CXCL13 (Abcam). CXCL13-expressing cells were identified with StreptABComplex/HRP (Dako) and 3,3-diaminobenzidine tetrahydrochloride solution (Sigma-Aldrich) to develop brown precipitates.

Embryos were fixed in 4% PFA overnight, embedded into OCT compound, and frozen. Frozen samples were sectioned at 5 μm thickness, dried up on slide glass, fixed in 4% PFA, and endogenous peroxidase activity was quenched by incubating the slides with 0.3% hydrogen peroxide. The sections were subjected to antigen retrieval by microwaving in sodium citrate (0.01 M, pH 6.0) as the antigen unmasking solution and then incubated overnight at 4 °C with the primary antibody. Antibodies used for this experiment are as follows: PCNA (PC 10, monoclonal, Sigma-Aldrich, St Louis, MO, USA), p27 (mouse monoclonal, Cell Signaling Technology, Danvers, MA, USA), activated caspase-3 (Cell Signaling Technology), BrdU (Bu20a, mouse monoclonal, Cell Signaling Technology), Nestin (clone 401, monoclonal, Merck Millipore), Hes5 (AB5708, polyclonal, Merck Millipore), and CDKN2A/p16INK4a (2D9A12, monoclonal, abcam).
Immunostaining with the mouse monoclonal antibody was carried out using an ABC kit (Vector Laboratories, Burlingame, CA, USA). After incubation with antibodies, sections were washed three times in PBS and then incubated with the appropriate biotinylated secondary antibody (DAKO and Vector Laboratories) followed by StreptABComplex/HRP (DAKO and Vector Laboratories) following the manufacturer’s instructions.

This retrospective study was approved by the Ethics Committee of the Northern Ostrobothnia Hospital District, Finland (49/2010, 56/2010) and the Finnish National Supervisory Authority for Welfare and Health (6865/05.01.00.06/2010). The formalin-fixed, paraffin-embedded blocks from patients with primary OTSCC treated between 1981 and 2005 were retrieved from the surgical pathology archives of the University Hospital of Oulu (n = 67). Detailed patient data are presented in Table 1.
Immunostaining was performed on 5-µm thick tissue sections using Herpes Simplex Virus I Rabbit Polyclonal Antibody (1:2000, Cell Marque). Briefly, after dewaxing and hydration in graded alcohol solutions, the antigens were retrieved by microwaving for 10 min in Tris-EDTA buffer pH 9.0 and cooled for 20 min at room temperature. Endogenous peroxidase was blocked using peroxidase-blocking reagent (S2023, DAKO) for 10 min. After washing with phosphate-buffered saline (PBS), the sections were treated with normal serum (Vector Laboratories, Burlingame, CA) in 2% bovine serum albumin/phosphate-buffered saline (BSA/PBS) for 30 min and then incubated with streptavidin-biotinylated horseradish peroxidase (StreptABComplex/HRP, Dako). Reactions were developed by incubating the sections with 3,3′-diaminobenzidine tetrahydrochloride (DAB, Vector) and counterstained with Mayer’s haematoxylin.