Dako real antibody diluent

10 µm tissue slides were blocked and permeabilized with 5% goat serum and 0.2% Triton X-100 and subsequently stained with the rabbit-anti-CNN3 antibody at 1∶50 dilution in Dako REAL Antibody Diluent (Dako, Germany) for 24 h at 4°C. As secondary antibody, the goat-anti-rabbit-Cy3 (Dako) was used at 1∶200 dilution for 2 h at room temperature in Dako REAL Antibody Diluent (Dako). To visualize actin filaments, the tissue slides were incubated with Alexa Fluor 488 Phalloidin (Molecular Probes, Germany) at 1∶5000 dilution for 15 min at room temperature. Nuclei were stained with DAPI (Molecular Probes) for 15 min at room temperature.

Slides were deparaffinized, rehydrated, and immersed in phosphate buffered saline buffer (10 mM PO43-, 0.,9% NaCl, pH 7.,2). Tissue epitopes were demasked through revitalization in TRIS-EDTA retrieval solution (10 mM TRIS, 1 mM EDTA, pH 9.0) at 98 °C for 20 min in Dako PT Link (Dako, Glostrup, Denmark). The slides were subsequently incubated for 90 min at room temperature with primary mouse monoclonal antibody against AIF (AIF (E-1): sc-13116, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:500 in Dako REAL antibody diluent (Dako) and immunostained using anti-mouse/anti-rabbit secondary antibody (EnVision FLEX / HRP, Dako) for 30 min at room temperature. The reaction was visualized by diaminobenzidine substrate-chromogen solution (DAB, Dako) which was applied for 5 min. Ultimately, the slides were counterstained with hematoxylin. Non-neoplastic testicular tissue was used as a positive control and the same tissue without incubation in primary antibody represented the negative control.

Formalin fixed, paraffin embedded lung tissues were cut into 5 μm thick sections, stained with hematoxylin/eosin and evaluated by light microscopy. Immunohistochemical staining was performed to detect EGFP expressed in tumor cells. Slides were deparaffinized and rehydrated in phosphate buffered saline solution (10 mM, pH 7.2). The tissue epitopes were demasked using the automated water bath heating process in Dako PT Link (Dako, Glostrup, Denmark); the slides were incubated in pH 6.0 citrate retrieval buffer at 98°C for 20 minutes. The slides were subsequently incubated for 60 minutes at room temperature with the primary rabbit polyclonal antibody against GFP (Abcam, anti-GFP, ab290) diluted 1:500 in Dako REAL antibody diluent (Dako, Glostrup, Denmark) and immunostained using anti-mouse/anti-rabbit immuno-peroxidase polymer (EnVision FLEX/HRP, Dako, Glostrup, Denmark) for 30 minutes at room temperature, according to the manufacturer’s instructions. For visualization, the slides were reacted with diaminobenzidine substrate-chromogen solution (DAB, Dako, Glostrup, Denmark) for 5 minutes. Finally, the slides were counterstained with hematoxylin.

Slides were deparaffinized and rehydrated in phosphate buffered saline solution (10 mM, pH 7.2). The tissue epitopes were demasked using the automated water bath heating process in Dako PT Link (Dako, Glostrup, Denmark); the slides were incubated in TRIS-EDTA retrieval solution (10mM TRIS, 1mM EDTA pH 9.0) at 98°C for 20 minutes. The slides were subsequently incubated for 1 hour at room temperature with the primary mouse monoclonal antibody against PD-1 (Abcam, [NAT105]: AB52587) and rabbit monoclonal antibody against PD-L1 (Abcam [EPR1161(2)]: AB174838) diluted 1:100 in Dako REAL antibody diluent (Dako, Glostrup, Denmark) and immunostained using anti-mouse/anti-rabbit immuno-peroxidase polymer (EnVision FLEX/HRP, Dako, Glostrup, Denmark) for 30 minutes at room temperature, according to the manufacturer's instructions. Color reaction was developed with diaminobenzidine substrate-chromogen solution (DAB, Dako, Glostrup, Denmark) for 5 minutes. Finally, the slides were counterstained with haematoxylin, mounted and reacted for 5 minutes with diaminobenzidine substrate-chromogen solution (DAB, Dako, Glostrup, Denmark) for visualization. PD-1 and PD-L1 positivity of lymphocytes in the tonsil was used as a positive control, same tissue with omitting of the primary antibody served as negative control.

Immunohistochemical staining was performed using formalin-fixed paraffin-embedded tumor blocks. Briefly, 4-µm thick tissue sections were deparaffinized in xylene and hydrated by immersing them in a series of graded ethanol. Antigen retrieval was performed in a microwave by placing the sections in epitope retrieval solution (0.01 M citrate buffer, pH 6.0) for 20 minutes; endogenous peroxidase was inhibited by immersing the sections in 0.3% hydrogen peroxide for 10 minutes. Sections were then incubated with the primary mouse monoclonal antibody to BCAT1 (BD Biosciences) and mouse monoclonal antibody to human IDH1 R132H (Dianova) in Dako REAL antibody diluent (Dako). Staining to detect the bound antibody was evaluated by DAB.

Immunohistochemical staining was performed using formalin-fixed paraffin-embedded tumor blocks. Briefly, 4-μm-thick tissue sections were deparaffinized in xylene and hydrated by immersing them in a series of graded ethanols. Antigen retrieval was performed in a microwave by placing the sections in epitope retrieval solution (0.01 M citrate buffer, pH 6.0) for 20 minutes; endogenous peroxidase was inhibited by immersing the sections in 0.3% hydrogen peroxide for 10 minutes. Sections were then incubated with the primary mouse monoclonal antibody to BCAT1 (BD Biosciences), mouse monoclonal antibody to human IDH1 R132H (Dianova), mouse monoclonal antibody to human KI67 (UM800033, ORIGENE), goat polyclonal antibody to rat CD34 (R&D Systems), or mouse monoclonal antibody to human HIF-1 alpha (Thermo) in Dako REAL antibody diluent (Dako). Staining for the detection of the bound antibody was evaluated by DAB.