Fitc conjugated goat anti rabbit

Anti-integrin immunofluorescence staining was performed on cryosections of hPLAP-prestained osteochondral explants as described previously.^{31 (link),32 (link)} In brief, sections were fixed with ice-cold acetone for 2 min at −20°C and blocked with 5% normal goat serum (Vector Laboratories) in PBS supplemented with 0.2% Tween-20 for 30 min at RT. Cryosections were stained overnight at 4°C for collagen I (rabbit polyclonal; Rockland Immunochemicals), collagen II (rabbit polyclonal; Rockland Immunochemicals), α1 integrin (hamster clone Ha31/8; BD Biosciences), α2 integrin (mix of three rat clones; Emfret), and α5 integrin (rabbit polyclonal; Santa Cruz Biotechnology), or a combination of α2 integrin and collagen I or collagen II for co-immunofluorescence staining, followed by incubation with FITC-conjugated goat anti-rabbit (Sigma), Alexa 488-conjugated goat anti-rat (Molecular Probes), Texas Red-conjugated goat anti-hamster (Vector Laboratories), and Cy3-conjugated goat anti-rabbit (Jackson ImmunoResearch), either single or in mixture, for 1 h at RT. Stained sections were analyzed and pictures were taken with a microscope (Axioskop 2; Zeiss).

After 7 days of culture, S42 cells cultured in each well were rinsed twice with PBS and then fixed with formalin for 20 min at room temperature (25°C). After that, the cell/nanofiber complexes were rinsed three times with distilled water, further dehydrated using a series of graded ethanol (50, 70, 90 and 100%, for 15 min for each concentration of ethanol), and naturally dried. After the samples were sputter-coated with gold, they were observed under SEM.
Furthermore, to illustrate the effects of various fibrous mats on S42 cell maturity, immunofluorescent staining with S100 was conducted. After 7 days of culture, cells on different fibrous mats were fixed with formalin for 20 min, permeabilized in 0.1% Triton X-100 solution for 10 min, and blocked in 3% w/v BSA aqueous solution for 90 min. The cells were treated with an anti-S100 antibody produced in rabbits (diluted at 1:100, Sigma) for 2 h at room temperature. Secondary antibody labeling was performed with FITC-conjugated goat anti-rabbit (Sigma) at a dilution of 1:300 for 1 h. After further treatment with mounting medium with DAPI (Vector Laboratories, USA), the samples were characterized using confocal laser scanning microscopy (CLSM, Zeiss LSM700).