Alexa fluor coupled secondary antibody

Cells were seeded onto sterile glass coverslips in 6-well dishes. Coverslips were washed twice with PBS, fixed with 3.7% (w/v) formaldehyde, 200 mM HEPES pH 7.0 for 10 min, washed twice with and incubated for 10 min in DMEM/10 mM HEPES pH 7.0. After one wash in PBS permeabilization was carried out using 0.2% NP-40 in PBS for 4 min. Samples were blocked by washing twice and incubation for 15 min in blocking buffer (1% (w/v) BSA/PBS). Coverslips were incubated for 1 h at 37°C with primary antibodies in blocking buffer and washed three times in blocking buffer. Coverslips were then incubated for 1 h at room temperature with Alexa Fluor coupled secondary antibodies (Life Technologies) in blocking buffer and washed an additional three times in blocking buffer. After submerging in ddH₂O, cells were mounted onto glass slides using prolong gold antifade mountant with DAPI (Life Technologies) and visualized with a Nikon Eclipse Ti-S fluorescence microscope (Nikon) or a Zeiss LSM880 Airyscan Confocal Scanning microscope (ZEISS; Plan Apochromat X40 objective, NA 1.4). Images were processed using Adobe Photoshop or ZEISS Zen Software.

BMSCs and HUVECs were cultured in osteoinductive medium and endothelial basal medium, respectively, in the presence of HPS and NHPS scaffolds, as described above. After incubating for 7 days, cells were fixed with 4% paraformaldehyde solution and washed with PBS. Next, cells were permeabilized with 0.2% (v/v) Triton X-100 for 15 min and nonspecific binding was blocked with 10% goat serum solution (Invitrogen, US). Then, BMSCs with the primary antibodies OCN (ab13420, Abcam, 1:100), OPN (ab8448, Abcam, 1:100) and HUVECs with VEGF (ab115805, Abcam, 1:100) and CD31 (ab28364, Abcam, 1:100) were incubated for 1 h at room temperature. After primary antibody incubation, cells were washed with PBS and incubated with appropriate Alexa-Fluor-coupled secondary antibodies (Life Tech, USA, 1:400) for 1 h at room temperature. The images were captured using a fluorescent microscope (ZEISS, Axio, Germany).

Mice were euthanized with CO₂ and perfused with PBS. For cryostat sections, the tissue was fixed overnight with 4% PFA, cryoprotected in 30% sucrose and frozen at −80°C. Frozen tissue was cut sagittally in 40-μm-thick sections and stained with myelin basic protein (MBP)-specific (Dako, Glostrup, Denmark) and β-actin-specific (Biolegend, San Diego, CA) antibodies. Detection was accomplished using Alexa fluor-coupled secondary antibodies (Life Technologies, Karlsruhe, Germany). Sections were covered with Vectashield (Vector Laboratories, Burlingame, CA) and analyzed by confocal (Leica SP7, Leica, Wetzlar, Germany) microscope.

Cells were fixed for 15 min at RT in 4% PFA followed by permeabilization with 0.1% Triton X-100 in PBS for 10 min. After washing in PBS, cells were incubated with anti-VE-cadherin AB overnight at 4 °C (Santa Cruz Biotechnology, #SC-6458). Alexa-Fluor-coupled secondary antibodies (Invitrogen), were added to the samples for 1 h at room temperature and cells were counterstained with Hoechst 33342. Samples were mounted in DAKO fluorescent mounting medium. ImageJ software was used to define cell periphery manually, and obtain the total cell area and circularity.