Streptavidin horseradish peroxidase

Cell culture and transfection reagents were bought from Life Technologies (Carlsbad, CA, USA). Human recombinant TGF-β1 was from R&D Systems (Minneapolis, MN, USA). Monoclonal antibodies against E-cadherin and N-cadherin, Matrigel and Transwell of 0.8 μm were purchased from BD Biosciences (San Jose, CA, USA). Monoclonal antibodies against total-Smad2/3, phospho-Smad2 and Smad3, Vimentin, Snail and Slug were purchased from Cell Signaling (Beverly, MA, USA). Biotinylated L-PHA, E-PHA, WGA, GNA, ConA, DSA and Fluorescein Avidin D, R.T.U. Horseradish Peroxidase Streptavidin were products of Vector Laboratories (Burlingame, CA, USA). Dky X Rb IgG (H+L) Cy3, Gt Ms IgG FLUOR and swainsonine were purchased from Merck Millipore (Darmstadt, Germany). Rhodamine Phalloidin was from Cytoskeleton (Denver, CO, USA). Anti-GnT-V antibody was purchased from Abcam (Cambridge, MA, USA). Anti-GAPDH antibody, Anti-Histone1 and secondary antibodies conjugated with HRP were ordered from Kang-Chen Biotech (Shanghai, China).

The total proteins isolated from the A2780 cells and A2780-cp cells were analyzed via SDS-PAGE and lectin blotting. Briefly, the samples were separated via 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with TBST (150 mM NaCl, 10 mM Tris–HCl, and 0.05% v/v Tween 20, pH 7.5) containing 5% bovine serum albumin for 1 h at room temperature, the PVDF membranes were incubated with the biotinylated lectins (Vector Laboratories, Burlingame, CA, USA) from Lens culinaris (LCA), Canavalia ensiformis (ConA), Lycopersicon esculentum (LEL) and Sambucus nigra (SNA) for 2 h at room temperature. The membranes were subsequently incubated with horseradish peroxidase streptavidin (Vector Laboratories) for 30 min and detected with an ECL assay kit.

For YKL40 IHC evaluation, heat-induced antigen retrieval was performed using Epitope Retrieval Solution pH 9 (Leica Microsystems, Wetzlar, Germany), as previously described. Subsequently, endogenous peroxidase was blocked for 10 min, with 30% hydrogen peroxide in methanol. To decrease background signal due to endogenous avidin, biotin, or biotin-binding proteins, a sequential incubation with avidin block (BioLegend, San Diego, CA, USA) and biotin block (BioLegend, San Diego, CA, USA) (10 min each) was performed. Tissues were then incubated for 10 min with tissue blocker UltraVision Protein Block (Thermo Fisher Scientific, Waltham, MA, USA). Incubation with the YKL40 primary antibody [(AF2599, R&D Systems, Minneapolis, MN, USA); dilution 1:300] was carried out overnight at 4 °C. The primary antibody was omitted in the slides used as negative control. Tissue sections were further incubated for 10 min with a biotinylated secondary antibody, polyclonal rabbit anti-goat immunoglobulins (Dako, Glostrup, Denmark; dilution 1:200), and subsequently with ready-to-use (R.T.U.) horseradish peroxidase streptavidin (Vector, Newark, CA, USA) for 30 min. Human tonsil was used as positive control.

Immunohistochemical analysis of osteocalcin was performed on paraffin‐embedded femur tissues (5 µm). Briefly, sections were deparaffinized and blocked with 10% goat serum for 2 hours at 4°C, followed by incubation with monoclonal antiosteocalcin antibody (1:500; ab93876; Abcam, Cambridge, MA, USA) overnight at 4°C. Thereafter, sections were incubated in 0.3% H₂O₂ (Merck) for 15 min, blocked with Avidin/Biotin Blocking Kit (15 min/15 min; Biozol, Bayern, Germany) and incubated with secondary antibody (biotynliated antirabbit, 1:800; BA‐1000; Vector Laboratories, Burlingame, CA, USA) for 2 hours at 4°C. Afterwards, sections were incubated with horseradish peroxidase streptavidin (1:300; SA‐5004; Vector Laboratories) for 60 min at 4°C and stained with ImmPACT DAB Peroxidase Substrate Kit (Vector Laboratories) for 3 min. Counterstaining was done with Hematoxylin Gill's Formula (Vector Laboratories) for 5 min; dehydration slides were mounted with Eukitt Mounting Media (R. Langenbrick, Emmendingen, Germany). Sections were visualized under a microscope (Olympus, Hamburg, Germany) using 400 × magnification. Sections without primary or secondary antibody were also included. Cells positive for osteocalcin stained brown.

Brains were dissected out, fixed in ethanol (60%), acetic acid (10%), and chloroform (30%), and included in paraffin. Deparaffinized tissue sections (20 μm) were incubated with 0.1% Triton X-100 (Sigma Aldrich, Cat# 93443; lot n°: BCBN7646V) for 15 min and then with hydrogen peroxide (3%) for 10 min. Slices were incubated for 1 h with 10% Normal Horse Serum (Sigma Aldrich, Cat# S-2000; lot n°: ZB0929), and successively for 30 min with monoclonal mouse anti-TH antibody in 2% Normal Horse Serum (1:100; Sigma Aldrich, Cat# T1299 RRID:AB_477560; lot n°: 015M4759V). Samples were then incubated for 10 min with secondary biotin-coupled anti-mouse antibody (1:400; Vector Laboratories, Cat# BA-2000; lot n°: Y0907), followed by exposure to Horseradish Peroxidase Streptavidin for 5 min (1:100; Vector Laboratories, Cat# SA-5004; lot n°: ZC1115). 3,3-Diaminobenzidine tetrachloride (Sigma–Aldrich, Cat# D4293; lot n°: SLBJ3609V) was used for detection. Negative control was performed without incubation with primary antibody.

Immunostaining was performed on paraffin slides of stomachs as described previously (7). Reagents used to detect staining were anti-Ki67 1:600, anti-CD163 1:400 (both rabbit anti-mouse antibodies from Abcam), biotinylated anti-rabbit secondary antibody, horseradish-peroxidase streptavidin (Vector Laboratories), 3’-diaminobenzidine (Sigma-Aldrich) and hematoxylin.