Alexa fluor 594 conjugated goat anti rabbit antibody

Cells were treated with 3 µM FMC for 12 h and processed for immunofluorescence using a monoclonal antibody against α-tubulin.
Briefly, after treatments cells were pelleted by centrifugation at 500 × g for 10 min, washed with PBS and adhered by cytocentrifugation on microscope slides. Adherent cells were fixed for 10 min at room temperature with 3% paraformaldehyde, washed once with PBS for 5 min, then 0.1 M glycine for 5 min at room temperature, followed with PBS, permeabilized (0.25% Triton X-100), washed with PBS and blocked with 5% BSA and 5% normal goat serum in PBS containing 0.025% Triton X-100 for 1 h. After washing twice with PBS, cells were incubated with 1% BSA in PBS containing 0.025% Triton X-100 containing anti-α-tubulin monoclonal antibody (#2125, Cell Signaling Technology, 1:100 dilution) overnight at 4 • C. After washing with PBS, cells were incubated with Alexa Fluor 594-conjugated goat anti-rabbit antibody (Invitrogen, 1:1,000 dilution) in the dark for 90 min. Then, cells were rinsed with PBS and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) containing DAPI (4 ′ ,6-diamidino-2-phenylindole, 1.5 µg/mL). Cellular microtubules images were obtained by using an inverted Nikon Eclipse 80i microscope with a 40x objective.

Immunofluorescence analyses of LR-MSCs and lung tissues were performed as described previously [32] . The following primary antibodies were employed: rabbit anti-Wnt10a, rat anti-Sca-1, mouse anti-α-smooth muscle actin (α-SMA), and rabbit anti-collagen I. Alexa Fluor 594-conjugated goat anti-rabbit antibody, Alexa Fluor 488-conjugated goat anti-mouse antibody, and Alexa Fluor 647-conjugated goat anti-rat antibody (all from Invitrogen) were used as secondary antibodies. Nuclei were stained with 2 mg/ml 4′,6-diamidino-2phenylindole (Sigma). Images were acquired using a confocal fluorescence microscope (Olympus, Tokyo, Japan).