Avidin biotin complex kit

Immunohistochemical staining was performed on 5-μm paraffin sections. After the sections were deparaffinized, heat-induced epitope retrieval was performed by immersing the slides in 0.01 M sodium citrate buffer (pH 6.0). To block endogenous peroxidase activity and the nonspecific binding of antibodies, the sections were preincubated for 1 h at room temperature in 0.1 M phosphate-buffered saline containing 10% normal goat serum and 0.3% H2O2. The sections were incubated with one of two primary antibodies: rabbit polyclonal anti-von Willebrand factor (vWF) antibody (1:100; Abcam, Cambridge, MA, USA) or rabbit polyclonal anti-vascular endothelial factor (VEGF) antibody (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The sections were treated with biotinylated goat antirabbit immunoglobulin G (IgG; 1:200, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). An avidin–biotin complex kit (Vector Laboratories, Inc., Burlingame, CA, USA) and a diaminobenzidine substrate kit (Vector Laboratories, Inc.) were used to visualize the brown reaction product, following the manufacturer’s recommendations. Vascular density was determined through immunohistochemical staining for vWF. Microvessel density was determined by counting the number of vessels positive for vWF staining in at least four random lung fields at 400× magnification in an unbiased manner [20 (link)].

Mice were anesthetized and perfused intracardially with phosphate-buffered saline (PBS, pH 7.2) for 30 s, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH 7.2. Brains were removed, cryoprotected in 30% sucrose at 4°C, and sectioned at 40 μm using a freezing microtome. Free-floating sections were in 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min and preblocked in 4% normal goat serum in PBS, 0.1% Triton X, and then incubated with mEM48 antibody at 4°C for 48 h. The immunoreactive product was visualized with the Avidin–Biotin Complex kit (Vector ABC Elite, Burlingame, CA, USA).
Cultured cells were fixed with 4% paraformaldehyde for 15 min, and then blocked for 1 h with 3% BSA and 0.2% triton X-100 in 1X PBS. Cells were incubated overnight with primary antibodies in 3% BSA in 1X PBS. The nucleus was visualized using Hoechst staining (Molecular Probes) at a dilution of 1:5000. Fluorescent images were obtained using a Zeiss microscope (Axiovert 200 MOT) with a digital camera (Hamamatsu Orca-100) and OpenLAB software (Improvision Inc).

Immunohistochemical staining was performed on 5-μm paraffin sections. After the paraffin sections were deparaffinized, heat-induced epitope retrieval was performed by immersing the sections in 0.01 M sodium citrate buffer (pH 6.0). To block endogenous peroxidase activity and the nonspecific binding of antibodies, the sections were preincubated for 1 h at room temperature in 0.1 M phosphate-buffered saline containing 10% normal goat serum and 0.3% H₂O₂. The sections were incubated with rabbit polyclonal anti-vWF antibodies (1:200, GTX60934; GeneTex, Irvine, CA, USA) for 20 h at 4 °C and biotinylated goat anti-rabbit IgG (1:200, Jackson ImmunoResesarch Laboratories, West Grove, PA, USA) for 1 h at 37 °C. The avidin–biotin complex kit (Vector Laboratories, Newark, CA, USA) and diaminobenzidine substrate kit (Vector Laboratories) were used to visualize the brown reaction product according to the manufacturer’s recommendations. All immunostained sections were viewed and photographed using an Olympus BX43 microscope. Digital images of each section were captured and quantified by counting the positive stained vessels in five randomly selected fields per section at × 400 magnification.

To determine the presence of glycan epitopes on primary monocytes and THP-1 cells, cell surface proteins on apical cell membranes were biotinylated using the Cell Surface Protein Isolation kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The biotinylation efficiency was confirmed by Western blotting using the Avidin-Biotin Complex Kit (Vector Laboratories, Newark, CA, USA).

Immunohistochemistry was performed using a previously described method (17 (link)). Briefly, 3 μm sections were deparaffinized in xylene and rehydrated in graded alcohol. After quenching endogenous peroxidase activity and blocking non-specific binding, the sections were incubated with a mouse monoclonal antibody against CD177 or F4/80 (Santa Cruz, CA, USA) overnight at 4°C and then incubated with a biotinylated secondary antibody (Zymed Laboratories, Carlsbad, CA, USA) at room temperature for 20 min. Finally, the slides were incubated with reagents from the Avidin-Biotin Complex Kit (Vector Laboratories, Burlingame, CA, USA) and a 3,3'-diaminobenzidine kit (Tiangen, Sichuan, China) according to the manufacturer's instructions. Images were captured with an Olympus CX43 microscope and CellSens Entry software.

The fixed brains were embedded in paraffin, and subsequently cut in sections of 5 µm. After deparaffinization with xylene and rehydration through graded ethanol series, the slides were cooked for 20 min in citrate buffer for antigen retrieval. Slides were then stained overnight at 4°C with anti-amyloid-β antibody (1:1000; Abcam ab2539) followed by a 1 hr room temperature incubation with biotinylated secondary antibody (1:300; Dako E0431). Immunodetection was visualized using an Avidin-Biotin Complex kit (Vector Laboratories, UK), and sections were counterstained with haematoxylin before mounting. The slides were digitized with an automatic bright field microscope (Philips Ultra Fast Scanner, Philips, the Netherlands) and assessed by one examiner (LPM) for positivity for amyloid-β.