Dab substrate chromogen system

Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue specimens. Briefly, 5 μm tissue sections were deparaffinized in Histoclear (Brunschwig, Basel, Switzerland) and rehydrated in descending concentrations of ethanol. Antigen retrieval was performed using citrate buffer, pH 6.0 (DAKO, Glostrup, Denmark) for 30 min at 98 °C. Endogenous peroxidases were blocked by incubation with 0.9% hydrogen peroxide for 15 min at room temperature (RT), blocking was performed using 3% bovine serum albumin for 1 h at RT in a wet chamber. Samples were stained for 1 h at RT with primary antibody F4/80 (clone D2S9R, Cell Signaling, #70076S), and for 1 h at RT with HRP-labeled secondary antibody. Antibody binding was visualized using a liquid DAB+ substrate chromogen system (DAKO). Then, samples were counterstained with hematoxylin and dehydrated in ascending ethanol solution and Histoclear.

IHC was performed on paraffin-embedded sections. The sections were deparaffinized in xylene and hydrated with decreasing concentrations of ethanol (100, 90, 80, 75%) for 3 min each time and microwaved-heated in sodium citrate buffer for antigen retrieval. Then, the sections were blocked in 5% BSA and incubated with anti-CD8 rabbit polyclonal antibody (1:1000, Abcam, UK) at 4 °C overnight. Next, the sections were treated with horseradish peroxidase (HRP)‑conjugated rabbit secondary antibody (1:200; ProteinTech Group, Inc., Wuhan, China) for 60 min at room temperature; then, 3,3′‑diaminobenzidine development (DAB Substrate Chromogen System; Dako, Denmark) and hematoxylin staining were performed. The sections were fixed and images were obtained with an inverted microscope (Olympus IX71, Japan).

For immunohistochemistry, paraffin blocks were cut into 4 μm sections, deparaffinized in xylene, and rehydrated in graded ethanol solutions. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 20 min, washed with phosphate‐buffered saline (PBS; 0.01 m, pH 7.4) three times for 5 min, and incubated with blocking buffer at 37 °C for 60 min. Tissue sections were incubated overnight at 4 °C with anti‐Desmin (1 : 200, ab15200; Abcam, Guangdong, China), anti‐α‐SMA (1 : 200, ab7817; Abcam), anti‐CK7 (1 : 8000, ab181598; Abcam), anti‐CK19 (1 : 200, ab7755, Abcam), or anti‐EpCAM (1 : 200, ab71916; Abcam) antibodies, and then with horseradish peroxidase (HRP)‐conjugated rabbit anti‐mouse IgG (A9044; 1 : 200) or anti‐rabbit IgG (A0545; 1 : 200) secondary antibodies for 1 h at 37 °C. HRP‐conjugated secondary binding was visualized using a DAB⁺ substrate chromogen system (Dako, Beijing, China). Nuclei were counterstained with hematoxylin. Bright‐field images were captured using an optical microscope (BX53; Olympus, Shanghai, China). Brown staining was considered positive. Positive areas were analyzed by using winroof software (V6.3, Mitani Corporation, Tokyo, Japan). The positive area (%) per high‐power field (200×) was calculated as follows: expression = positive area/total area × 100%.

For pre-treatment, TMA sections (2 μm) were incubated in antigen retrieval solution with pH 6 or 9 (PT Link, Dako) at 95°C for deparaffinization, rehydration and epitope retrieval. FFPE slides were then treated with EnVision^TM Flex peroxidase blocking reagent (Dako) for 5 min to block endogenous peroxidase activity. Immunostaining was performed with antibodies specific for ARID1A (1:250, D2A8U, Cell Signaling), EZH2 (1:50, 6A10, Leica) and H3K27me3 (1:500, C36B11, Cell Signaling). Subsequently, tissue sections were incubated with a HRP-conjugated secondary reagent (Dako) for 15 min. The peroxidase reaction was visualized with the DAB+ Substrate Chromogen System (Dako). The sections were then counterstained using Mayer`s haematoxylin. Nuclear expression of ARID1A, EZH2 and H3K27me3 was assessed by two experienced pathologists (RK and NTG) according to an adapted immunoreactive score (IRS) developed by Remmele and Stegner (1987) [31 (link)].

Immunohistochemistry for all antibodies except aggrecan was performed using standard diagnostic procedures using the Omnis autostainer (Dako, Agilent). Depending on the type of antibody, antigen retrieval was carried out in either Tris-EDTA pH 9.0 or citrate pH 6.0. Details of antibodies dilutions, use of linkers, and other information are listed in Supplementary Table 2 (Supplemental Digital Content 2, http://links.lww.com/PAS/B163). For aggrecan, 4-µm deparaffinized FFPE sections were rehydrated and endogenous peroxidase blocked. Antigen retrieval was performed with citrate, pH 6.0. Tissue sections were then incubated with antibody overnight at 4°C in 1:16,000 dilution. Detection was applied using immunologic Poly-HRP-GAM/R/IgG (VWR, DVPO110HRP) and Dako liquid DAB+ Substrate Chromogen System (K3468) as described.14 (link) Slides were counterstained with hematoxylin. Round cell sarcomas with molecularly proven EWSR1-NFATC2 rearrangement, previously described as index cases, located in the femur of 16 years old male (L1857) and the humerus of 36 years old male (L1231) were used as control.4 (link)

Femur specimens from mice were fixed with 4% paraformaldehyde, decalcified with 10% EDTA and embedded in paraffin. Sections were deparaffinized, treated with 3% H₂O₂ to inhibit endogenous peroxidase activity, blocked with goat serum, and then incubated for overnight at 4°C with 1:10 dilution of the rabbit polyclonal anti-mouse sclerostin antibody (Sigma-aldrich) and 1:50 dilution of the rabbit polyclonal anti-mouse RANKL antibody (abcam). Sections then were incubated for 30 minute at room temperature with a 1:300 dilution biotinylated anti-rabbit IgG secondary antibody (Vector). Sections were further incubated for 30 minutes with a 1:300 dilution of peroxidase-conjugated streptavidin (DAKO) in 2% goat serum and developed with a DAB substrate-chromogen system (Dako) for up to 5 minutes. We confirmed no non-specific immunostaining with a rabbit control IgG as a primary antibody.