Alexa fluor 546 goat anti rat igg

The cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Fixed cultures were washed with PBS, permeabilized with 0.2% Triton X-100 in PBS for 3 min, and blocked for 1 h with 5% bovine serum albumin (BSA). Next, the cells were incubated with anti-BrdU (Sigma, 1:500), polyclonal anti-fibronectin (Sigma, 1:400) and anti-laminin (Sigma, 1:50), anti-Sox2 (Millipore, 1:100), anti-CD133 (Millipore, 1:20) antibodies or double-stained with polyclonal anti-GFAP (DAKP, 1:500) and monoclonal anti-Nestin (BD Biosciences 1:50) antibodies overnight at 4°C. Incubation with specific secondary Alexa Fluor 546 goat anti-mouse IgG (Molecular Probes 1:800), Alexa Fluor 488 goat anti-rabbit (Molecular Probes 1:400) or Alexa Fluor 546 goat anti-rat IgG (Molecular Probes 1:500) proceeded for 1 h at room temperature. Then, the cells were washed with PBS, stained with DAPI (Sigma), washed again and mounted. For negative controls, cells received similar treatment but the primary antibodies were omitted. The slides were observed in a Nikon TE 2000 inverted microscope. Images were captured using a CoolSNAP-Pro (Media Cybernetics) digital camera.

To detect blood vessels and macrophages in the connective tissue of tooth extraction sockets, rat anti-mouse CD31 monoclonal antibody (ab56299; Abcam, Cambridge, MA; 1:100 dilution), rat anti-mouse F4/80 monoclonal antibody (ab16911; Abcam; 1:50 dilution), and rabbit anti-mouse CD206 polyclonal antibody (ab64693; Abcam; 1:50 dilution) were used as primary antibodies. Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 546 goat anti-rat IgG (Invitrogen, Carlsbad, CA; 1:200 dilution) were used as secondary antibodies. Sections were mounted with VECTASHIELD Antifade Mounting Medium with DAPI (H-1200; Vector Laboratories, Burlingame, CA). Stained sections were photomicrographed by fluorescence microscopy (Axio Scope A1; Zeiss) and were histomorphometrically analyzed with ZEN2 software and NIH ImageJ to yield the following parameters: the number of blood vessels [blood vessels (#/mm²)]; vessel surface area in the AOIs of soft tissue in the tooth extraction sockets [vessel density (%)] (AOI, 200 × 1000 μm); F4/80⁺CD206⁻ macrophages [F4/80⁺CD206⁻ cells]; and F4/80⁺CD206⁺ macrophages [F4/80⁺CD206⁺ cells] in the AOIs of soft tissue in the extraction sockets (AOI, 200 × 1000 μm).