3 3 diaminobenzidine chromogen

Joint tissues were first incubated with primary antibodies against IL-1β (R & D Systems, Minneapolis, MN, USA), IL-6 (R & D Systems), TNF-α (R & D Systems), IL-17 (R & D Systems), and RANK ligand (RANKL; R & D Systems) overnight at 4°C. Samples were incubated with a biotinylated secondary antibody, followed by incubation with a streptavidin–peroxidase complex for 1 h. Samples were then developed using chromogen 3,3′-diaminobenzidine (Thermo Scientific, Rockford, IL, USA). The sections were examined using a photomicroscope (Olympus, Tokyo, Japan).

Joint tissues were first incubated with primary antibodies against IL-1β (R&D Systems, Minneapolis, MN, USA), IL-6 (R&D Systems), TNF-α (R&D Systems), IL-17 (R&D Systems), RIPK1 (Cell Signaling Technology, Danvers, MA, USA), RIPK3 (Cell Signaling), pMLKL (Cell Signaling), tartrate-resistant acid phosphatase (TRAP; R&D Systems), receptor activator of nuclear factor kappa-B (RANK; R&D Systems) and RANK ligand (RANKL; R&D Systems) overnight at 4 °C. Samples were incubated with a biotinylated secondary antibody, followed by incubation with a streptavidin–peroxidase complex for 1 h. Samples were then developed using chromogen 3,3′-diaminobenzidine (Thermo Scientific, Rockford, IL, USA). The sections were examined under a photomicroscope (Olympus, Tokyo, Japan). The number of positive cells was counted using Adobe Photoshop software (Adobe, USA) on high-power digital image (magnification: 400×). Positive cells were enumerated visually by three individuals, and the mean values were calculated.

After deparaffinization and dehydration, slides were treated by blocking endogenous peroxidase with 0.3% methanol (Merck)/30% H₂O₂ (Merck) for 10 min in the dark. For periostin staining, sections were pretreated with pepsin for 20 min at 37 °C. Subsequently, sections were pre-blocked with 1 × tris-buffered saline (Merck)/4% bovine serum albumin (Merck) for 1 h at room temperature and incubated in a humid chamber overnight at 4 °C with a rabbit polyclonal antibody against periostin with a concentration of 1:300 (ab14041, Abcam, Cambridge, UK). For the detection of osteopontin and osteocalcin, no pre-treatment of sections was needed. Sections were immediately incubated with a rabbit polyclonal antibody against osteopontin with a concentration of 1:300 for 1 h at room temperature (ab8448, Abcam) and with a mouse monoclonal antibody against osteocalcin with a concentration of 1:1,200 overnight in a humid chamber at 4 °C (ab13418, Abcam). Afterwards, sections were rinsed and incubated at room temperature with a goat anti-rabbit IgG-HRP secondary antibody or a goat anti-mouse IgG-HRP secondary antibody (Dako, Hamburg, Germany) for 30 min. Peroxidase activity was visualized by incubation with 3,3-diaminobenzidine chromogen (Thermo Fisher Scientific, Dreieich, Germany). Finally, sections were rinsed and counterstained for 30 s with Mayer’s hematoxylin (Merck).

Tissues in the paraffin blocks were cut to a thickness of 4 μm and subjected to deparaffinization with xylene and dehydration with ethanol. For antigen retrieval, tissue sections were boiled in sodium citrate buffer (10 mM sodium citrate and 0.05% Tween 20, pH 6.0) for 10 min on a hot plate and then incubated at 25 °C for 20 min. After washing with PBS, the tissue sections were quenched in 3% H₂O₂ in PBS and incubated in blocking buffer (PBS, 1% BSA, and 0.5% Triton X-100) at 25 °C for 30 min. After washing with PBS, the tissue sections were incubated overnight with the primary antibody in a humid chamber at 4 °C. Tissue sections were washed with PBS and incubated with an HRP-conjugated secondary antibody (Invitrogen) in a humid chamber at 25 °C for 1 h. Tissue sections were washed with PBS, stained with 3,3-diaminobenzidine chromogen (Thermo) for 5 min at 25 °C, washed with distilled water, and counterstained with hematoxylin (DAKO, Carpinteria, CA, USA). Stained tissue sections were mounted using Permount (Thermo Fisher Scientific), and the stained images were examined by light microscopy. IHC staining was performed using the IHC profiler plugin of the ImageJ software [46 (link)].

Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded tissue sections 3μm thin. Slides were placed for 20 minutes in 10mM sodium citrate buffer, pH 6.5 heat at 97°C in order to unmask antigens. To reduce nonspecific background staining due to endogenous peroxidase, specimens were incubated in UltraVision Hydrogen Peroxide Block (Thermo Scientific) for 10 minutes and after they were washed in Tris Buffer, for 5 minutes in Ultra Vision Protein Block (Thermo Scientific) to block nonspecific background staining. Sections were incubated for 60 minutes at room temperature with EF-1 alfa 2 (D-15 santa cruz-68481) to detect eEF1A2:sc-68481 mixed with EF-1 alfa1 (CBP-KK1): sc-21758 used at 1:300 dilution and after washed in Tris Buffer, for 30 minutes at room temperature with goat anti-rabbit IgG,F(ab’)2 –HRP: santa cruz-3837 used at 1:300 dilution. Finally, sections were incubated for 10 minutes with 3,3’ Diaminobenzidine chromogen (DAB Quanto–Thermo Scientific) and hematoxylin to nuclear counter stain.
As positive controls, we used staining intensity of eEF1A2 in normal breast parenchyma (external positive control) and staining intensity of eEF1A2 in normal breast parenchyma, if present in tumor tissue section (internal control).