Fitc conjugated goat anti rabbit antibody

Osteoclast precursors resulting from canine bone marrow cells (2 × 10⁵ cells) were cultured in 8-well culture slide (Iwaki, Tokyo, Japan) in 400 μL of DMEM, 10% FBS with OC differentiation factors. Cells were incubated with 10 μg/mL of Tetramethylrhodamine (TRITC)-labeled NaPPS (Arthropharm, New South Wales, Australia) for 24 h. After fixation and blocking, cells were incubated with primary anti-human c-Jun (HT-9) rabbit polyclonal antibody (Santa Cruz Biotechnology, Dallas, Texas, USA) (1:100 dilution) in 1% normal goat serum followed by incubation with FITC-conjugated goat anti-rabbit antibody (Sigma) (1:100 dilution) in 1% normal goat serum. The OC were observed for detecting the colocalization patterns of NaPPS with transcribed gene (c-Jun) under a laser scanning confocal microscope.

HCC cells were seeded into 24-well culture plate with a glass coverslip over each well and allowed to attach overnight. The cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min and then permeabilized with 0.25% Triton X-100 (Sigma) for 10 min. After blocked with 4% bovine serum albumin, the cells were incubated overnight at 4°C with rabbit anti-Fibronectin or anti-E-cadherin antibody (Cell Signaling Technology, Danvers, MA, USA). On the following day, the cells were incubated with secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (Sigma-Aldrich, Poole, UK) for 1 h. HCC cells were then incubated with 4’,6-diamidin-2-phenylindole (DAPI; Life Technologies, Carlsbad, CA, USA) for 15 min to stain the nuclei. Images of the cells were acquired using a fluorescence microscope (FV1000; Olympus, Tokyo, Japan).

Immunophenotypic analysis was performed by staining 2 × 10⁵ MNCs, purified and expanded CD133⁺ cells per tube. MNCs and purified cells were analyzed after isolation, whereas expanded cells were analyzed at passage 3 or 4. Anti-mouse IgG1 antibodies conjugated with phycoerythrin (PE), fluorescein isothiocyanate (FITC), allophycocyanin (APC), and peridinin chlorophyll protein (PerCP) (all from BD Pharmingen™ San Jose, CA, USA) were used as isotype controls. The cells were incubated with the following monoclonal antibodies to determine their typical cell surface epitope profiles: anti-CD14, anti-CD31, anti-CD34, and anti-CD45 (all from BD Pharmingen™ San Jose, CA, USA), anti-CD105 (eBioscience Inc., San Diego, CA, USA), and anti-CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany). For intracellular detection of vWF, cells were permeabilized using FIX & PERM cell permeabilization reagents (Caltag, Carlsbad, CA, USA) and further incubated with an isotype-specific FITC-conjugated goat anti-rabbit antibody (Sigma-Aldrich, São Paulo, Brazil). Their viability was assessed by 7-AAD (BD Pharmingen) staining. The data for cell staining were acquired using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).