Envision flex detection system

In this study, the expression of FOXP3, CD4, and CD8 was evaluated in 84 of the FFPE samples included using a Dako Autostainer Link 48 and the Dako EnVision™ FLEX detection system (Dako, Burlington, Canada). Briefly, sections were dried and loaded into the PT Link instrument where the antigen retrieval/dewaxing process took place. The sections were transferred to the Autostainer Link 48 instrument where the endogenous peroxidase activity was quenched with peroxidase blocking reagent for 10 minutes. Immunostaining was carried out with Dako FLEX Ready to-Use format for CD4 (Clone 4B12, Dako) and CD8 (Clone C8/144B, Dako), and with a primary antibody diluted at 1:300 with Dako Antibody Diluent (Dako) for FOXP3 (Clone 236A/E7, Abcam, Cambridge, MA, USA). After incubation with the primary antibody a detection system chromogen (3,3’-diaminobenzidine, DAB) was used. Finally, the sections were washed, lightly counterstained with hematoxilin, dehydrated, and mounted. Normal human tonsil tissue was used as a positive control for the three antibodies, and negative controls were included by omitting the primary antibody.

Deparaffinized sections were incubated with antibodies to CD44 (ab157107, Abcam, Cambridge, UK); STRO-1 (ab57834, Abcam), osteocalcin (OC, ab198228, Abcam), osteopontin (OPN, ab218237, Abcam), dentin sialophosphoprotein (DSPP, ab216892, Abcam), alkaline phosphatase (ALP, ab216892, Abcam), pan-cytokeratin (pan-CK, CF190321, TrueMAB™ Thermo Fisher Scientific), vimentin (Vim, MA5-11883, Thermo Fisher Scientific). Antigen demasking was performed before immunohistochemical staining using low pH EnVison FLEX Target Retrieval Solution (Dako, Glostrup, Denmark A/S) at 97 °C for 20 min. Endogenous peroxidase and host IgG were blocked. Primary antibodies were diluted according to the manufacturer’s recommendations. Incubation was carried out for 12 h at 4 °C. An EnVision FLEX detection system (Dako, Glostrup, Denmark A/S) with 2,3-diaminobenzidine DAB chromogen (DAB Chromogen Solution, Dako) was used for imaging. The nuclei were counterstained with hematoxylin. We used incubation without primary antibodies as a negative control.

Tissue sections were fixed with 4% phosphate-buffered formaldehyde, embedded in paraffin and cut into 4.5-μm-thick sections. Immunohistochemistry was performed using Dako Autostainer LINK 48 using Envision FLEX+ detection system (Dako, Glostrup, Denmark). Briefly, deparaffinized sections were subjected to antigen retrieval by high-pressure cooking and DIVA antigen retrieval pH 6.2, followed by blocking with 3% hydrogen peroxide and incubation with primary antibody against anti-Ki67 (monoclonal mouse anti-human Ki-67 antigen clone MIB-1. M7240 (Dako) and anti-sortilin antibody (polyclonal rabbit anti-sortilin, #AB16640, Abcam) at RT for 1 h. For signal amplification EnVision™ FLEX+ rabbit linker, SM805, (Dako) and EnVision™ FLEX+ mouse linker SM804 (Dako) was used, respectively. Further EnVision FLEX/HRP visualization reagent EnVision™ FLEX/HRP secondary antibody-coated polymer peroxidase complexes (#SM802, Dako), followed by DAB substrate/chromogen (Dako) was used. Slides were counterstained with Dako hematoxylin. Stained sections were scanned by Leica SCN400 scanner at 20× and evaluated by the automated image analysis Definiens Developer XD tissue studio program.

Immunohistochemistry on a tissue microarray, constructed as described previously,²⁰ was performed for the detection of CD2AP expression. In brief, the sections were dewaxed by incubation with dimethyl benzene at 45°C for 60 minutes and then immersed in distilled water. Endogenous peroxidase activity was inhibited by incubation in a 0.5% hydrogen peroxide bath for 10 minutes. After washing three times with 0.01 M phosphate‐buffered saline (PBS, pH 7.4), the slides were immersed in citrate antigen retrieval buffer (Zhongshan Golden Bridge Biotechnology, Beijing, China). After blocking with sheep serum for 30 minutes, the sections were incubated with anti‐CD2AP antibody (sc‐25272; Santa Cruz Biotechnology, Dallas, TX; 1:50 dilution; Figure S1B) in a humidified chamber at 24°C for 2 hours. After washing three times with PBS, a Dako EnVision FLEX detection system (Dako, Carpinteria, CA) was used for visualization, according to the manufacturer's instructions. The sections were counterstained with hematoxylin, dehydrated, and sealed with neutral gum.

Paraffin-embedded tissue samples of 5-µm sections were subjected to IHC staining. Tissue sections were deparaffinized and rehydrated in graded ethanol solutions. Then, the deparaffinized tissue specimens were boiled in 10 mM citrate buffer (pH 6.0) for 100 min for antigen retrieval. Subsequently, the endogenous peroxidase activity was blocked by incubating with 3% H₂O₂ solution. The tissue sections were then incubated with the antibody in a humidified chamber at 37 °C for 2 h (Table S3). After washing thrice with phosphate buffered saline (PBS), a Dako EnVision FLEX detection system (Dako, Carpinteria, CA, USA) was used for visualization, according to the manufacturer’s instructions. The sections were counterstained with hematoxylin, dehydrated, and sealed with neutral gum. IHC staining was evaluated by experienced pathologists using a semi-quantitative scoring system based on the intensity of staining and the percent of positively-stained cells. The staining intensity of specimens was scored as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The positive expression was scored as 0 (0%), 1 (<25%), 2 (25–50%), 3 (50–75%), and 4 (>75%) based on the percentage of positive stained cells. The final staining scores for all samples were obtained by multiplying the staining intensity.