Dako real detection system

Tumor specimens were fixed in 10% formalin overnight and embedded in paraffin. For immunohistochemistry, slides were deparaffinized and rehydrated. Antigen retrieval was enhanced by boiling in a steam pot at pH 6 in Dako target retrieval solution (Dako) for 15 min, followed by cooling for 30 min and washing in distilled water. Nonspecific binding was blocked by using the Linaris avidin/biotin blocking kit (Vector Laboratories) according to the manufacturer’s instructions. Slides were incubated with primary antibodies for 30 min, rinsed in PBS-T (PBS with 0.5% Tween 20), incubated for 20 min with the appropriate secondary antibody using the Dako REAL detection system (Dako), and rinsed in PBS-T. After blocking of endogenous peroxidase and incubation with streptavidin-horseradish peroxidase (HRP) (20 min at room temperature), slides were developed with 3-amino-9-ethylcarbazole (AEC) (Dako) and counterstained with hematoxylin. All antibodies were diluted in Dako antibody diluent, including anti-SMAD4 (dilution 1:50; Santa Cruz), Ki67 (dilution 1:1,000, Sigma), and CK19 (dilution 1:200, Abcam).

Immunohistochemistry staining was done on 0.5–1 µm slides of paraffin embedded RMS biopsies by using routine immune peroxidase techniques. For quality control reasons, tonsils were included as positive controls. Pretreatment of the sections is depending on the used antibody. Antibodies, pretreatment conditions and antibody dilutions are listed in Supplemental Table S5. Immunohistochemistry was performed as described^{63 (link)} using the chemicals and reagents listed below: antigen retrieval in Novocastra antigen retrieval solution ph6 or pH 9.0 (Leica, Wetzlar, Germany); blocking of endogenous peroxidase (DAKO blocking solution, DAKO, Hamburg, Germany); detection of bound antibodies by the immunoperoxidase/DAB-based DAKO REAL detection system (DAKO).

Immunohistochemistry for PTEN was performed using 4 μm thick slides and standard protocols as described earlier [12 (link)]. For immunohistochemical staining of PTEN, antigen retrieval was performed in sodium citrate buffer (Biogenex, Hamburg, Germany) followed by incubation with a PTEN antibody overnight at 4°C (1:50, 138G6; Cell Signaling Technology, Danvers, MA) and Dako REAL Detection System (Dako). Cytoplasmic staining was scored as negative expression (0), low expression (1), or high expression (2). For statistical analyses, expression was defined as either negative (0) or positive (1 and 2).

Immunohistochemistry was performed on serial 2 µm-thick tissue sections cut from formalin-fixed, paraffin-embedded tissue blocks. Staining was performed according to standardized protocols using the commercially available Dako REAL detection system (Dako, Santa Clara, USA) and the Vectastain Elite Kit (Vector Laboratories, Burlingame, USA). Briefly, all slides were deparaffinized in xylene and underwent a series of incubations in decreasing ethanol concentrations for rehydration. Antigen retrieval was performed using different treatments specific for each antibody, including steaming (PD-L1, Nectin-2) and microwaving (PVR, PD1 and TIGIT) in different buffer solutions, EDTA buffer pH 9.0 (PD-L1, Nectin-2), citrate buffer pH 6.0 (PD-1, PVR) or TRIS-based buffer pH 9.0 (TIGIT) for 20 min. Incubation with the primary antibody was carried out for 30 min at room temperature (PD-L1, Nectin-2, PVR, PD-1) or overnight for at least 16h at 4°C (TIGIT). Further, sections were counterstained with hematoxylin.

Formalin-fixed paraffin-embedded (FFPE) tissue samples were cut in 4-µm-thick sections. Immunohistochemical staining was performed using the Dako REAL™ Detection System (#K5001, Dako) and an automated immunostainer (AutostainerLink 48, Dako). The biotin–streptavidin technique was used. In short, sections were deparaffinized and intrinsic peroxidase activity was blocked by incubation with 5% H₂O₂ in phosphate-buffered saline (PBS) for 5 min. Primary antibodies were applied as listed in Supplementary Table 1. IHC was completed using species-specific biotinylated secondary anti-mouse or rabbit antibodies followed by incubation with streptavidin/peroxidase complex and the reaction product was developed with diaminobenzidine. For quantitative evaluation, sections were analysed at 100-fold magnification using a morphometric grid, and average counts per square millimeter were calculated and compared by statistical analysis.

CD4⁺, FOXP3⁺, and IL-17⁺ cells were detected in muscle biopsy samples by immunohistochemistry. Acetone-fixed, frozen sections were exposed to monoclonal antibodies against CD4 (Dako clone MT-310), FoxP3 (eBioscience, clone 236A/E7), or IL-17 (R&D systems Clone AF-317-NA) overnight at 4°C. Positive staining was revealed by peroxidase reaction (Dako real™ Detection System (K5001; Dako)). Double immunofluorescence was used to analyse FoxP3⁺ and CD4⁺ cell colocalisation. Detection of mouse monoclonal anti-FoxP3 and rabbit polyclonal anti-CD4 (Abcam®) antibodies was performed using Alexa Fluor (AF) 555 goat anti-mouse IgG (L+H) and AF 488 goat anti-rabbit IgG (L+H) secondary antibodies. For control purposes, a mouse IgG1 isotype control was included in the protocol. Stained muscle sections were analyzed with a Leica TCS-SP (UV) confocal microscope at the MSSM-Microscopy Shared Resource Facility (Pitie-Salpetriere Hospital, Paris).

Immunohistochemical staining was performed as previously described with a Dako Dako REAL™ Detection System (Zhu et al. 2013 (link)). In brief, 4-μm formalin-fixed paraffin sections were cut. After the antigen retrieval in citrate buffer, sections were blocked with 5 % normal goat serum at room temperature and stained with antibodies against GTSE1 (Proteintech Group, Inc., Chicago, IL, USA), followed by incubation with goat anti-Rabbit IgG-biotinylated secondary antibodies (Dakopatts, Glostrup, Denmark), and visualized by standard avidin-biotinylated peroxidase complex method. Staining intensity was evaluated by two investigators who were unaware of clinicopathological features of the patients. Dark brown staining was defined as positive, and no staining was defined as negative. The percentage of positive cells and the intensity of immunostaining were used to produce a weighted score for each case.