Real peroxidase blocking solution

Staining was performed on fresh frozen (FF) human tissues and xenograft tumors (cryosections, 4 µm). Air-dried sections were fixed with 4% formaldehyde for 5 min at RT and washed for 5 min three times with PBS. Sections were blocked for endogenous peroxidase for 10 min at RT using REAL Peroxidase-Blocking Solution (S2023, Dako, Oslo, Norway) and washed three times for 5 min with dH₂O. Sections were then incubated with blocking solution (TBS containing 10% goat serum, 5% BSA and glycine at 0.3 M final concentration) for 60 min. Slides were drained and incubated with primary antibodies anti-HER2 (2.65 μg/mL) or Oslo-2 mAb (5 μg/mL) diluted in protein blocking solution for 60 min at RT. Sections were washed three times for 5 min with TBST. Polink-2 Plus HRP Broad DAB detection Kit (D41-18, Golden Bridge International, Bothell, CA, USA) was used according to the manufacturer’s instructions to detect HER2 or p95HER2 target proteins.
The scoring of HER2 protein expression in the present study into 0 (negative), 1+ (weak), 2+ (moderate), and 3+ (strong) was performed according to international standard guidelines, as described in the Ventana anti-HER2/neu (4B5) interpretation guide (Ventana, AZ, USA).

Immunohistochemical analysis (IHC) was performed on serial sections 3 μm thick. Deparaffinized tissues were heated to expose the hidden antigens using Antigen Retriever containing citrate buffer, pH 6.0 (Sigma Aldrich, St. Louise, United States). Endogenous peroxidase was blocked with Real Peroxidase-Blocking Solution (Dako, Glostrup, Denmark). Samples were stained using primary antibodies to detect podoplanin (Origene Technologies, Rockville, United States); α-SMA (Sigma Aldrich); ET_AR (ThermoFisher Scientific) and ET_BR (Abcam, Cambridge, United Kingdom). A biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, United States) followed by R.T.U Vectastain Elite ABC Kit (Vector Laboratories) was applied to detect primary antibodies. All cases were revealed using DAB (Dako) as chromogen and finally counterstained with haematoxylin. Representative images were captured with a digital camera coupled to a brightfield microscope.

IHC detection of key EMT proteins was performed using the Dako Envision FLEX + system (Dako, Berlin, Germany). Paraffin sections were deparaffinized. Antigen retrieval was performed by heating the samples in citrate buffer (pH 6.0) in the microwave for 15 min, then returning the samples to room temperature, followed by washing with phosphate-buffered saline (PBS). The samples were blocked with the Dako REAL Peroxidase-Blocking Solution for 15 min. The slides were incubated at 4°C overnight with the rabbit antibodies for anti-N-cadherin, Rabbit ant-E-cadherin, anti-phospho-AKT (Ser473), anti-GSK3B (phospho-Tyr216), and anti-GSK3B (phospho-Ser9), followed by incubation (30 min) with the secondary antibody. Slides were stained with 3,3′-diaminobenzidine tetrahydrochloride (DAB) for 2 min. The percentages of cells that were positive for the markers were scored as follows: 0–5%, no positive cells; 1, <25% positive cells; 2, 25–50% positive cells; 3, 50–75% positive cells; 4, 75–100% positive cells. The staining intensity was scored as follows: 0, no positive cells; 1, weak staining; 2, moderate staining; and 3, strong staining. The immunohistochemical staining score was obtained by multiplying the percentage score by the intensity (0, 1, 2, 3, 4, 6, 8, 9, or 12). The X-tile v3.6.1 statistical package [27 (link)] was used to analyze the IHC assay results.

Sections (10 µm) from snap-frozen mouse brains fixed with acetone were blocked for 60 min (DAKO REAL Peroxidase-blocking solution, DAKO serum-free protein block) and incubated for 60 min with antibodies against adenovirus (Millipore), CD8α (clone 53–6.7, eBioscience), or CD4 (clone GK1.5, eBioscience) followed by the secondary antibodies rabbit anti-goat IgG or goat anti-rat IgG (Invitrogen). Sections were stained with DAB chromogen (DAKO) and hematoxylin (Sigma-Aldrich) and mounted with Pertex (Histolab). Sections were photographed on an Axio Observer D1 microscope with 40× magnification (Zeiss).

Tumours were excised from BALB/c athymic nude mice at the experimental endpoint and fixed in 10% (v/v) buffered formalin. Fixed-tumours were then paraffin-embedded and sectioned at 4 µm onto Superfrost Plus slides. Immunohistochemistry was carried out using the DAKO Autostainer Link 48. Sections underwent dewaxing, heat-induced antigen retrieval using DAKO Target Retrieval Solution (S1699) at 98 °C for 30 min, endogenous peroxidases were quenched by applying Dako Real Peroxidase Blocking solution (S2023) for 10 min, followed by Dako Serum Free Protein Block (X0909) for 30 min. Then, primary antibody incubation using NRBP1 or Ki-67 antibody was followed by the Dako Envision + System – HRP Labelled secondary antibody incubation system. Subsequently, sections were counterstained with Dako Automation Hematoxylin Histological Staining Reagent (S3301). For the analysis, 10 random fields of vision images per sample were taken with ImageScope viewer and quantified using the Fiji ImageJ software (Version 1.52).

For immunohistochemistry, paraffin-embedded sections were deparaffinized in ethanol, and endogenous peroxidase was inactivated in 3% hydrogen peroxide (DAKO REAL Peroxidase Blocking Solution (#2023)). Sections were blocked with 1% BSA/PBS before incubating overnight with anti-mouse Ki67 antibody (Abcam #ab1667 1:600) or anti-mouse MMP2 antibody (Abcam #ab37150 1:800). For hematoxylin and eosin (H and E) staining, sections were incubated for 30 sec in hematoxylin and for 10 min in 1% eosin. Briefly, histological views were digitalized, and metastases were outlined. Quantification of the average value staining intensity was measured with ImageJ Plugin IHC Toolbox (ImageJ software, NIH, MD, USA).