Alexa flour 594 conjugated goat anti rabbit igg

Cells were cultured on glass cover slips in 4-well plates 18 h prior to treatment with the anti-hTM4SF5 monoclonal antibody or control IgG (10 μg/ml). After treatment with the antibody for the indicated time periods, the cells were fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton X-100, and stained with the anti-E-cadherin antibody (rabbit polyclonal Ab, Santa Cruz Biotechnology, sc-7870) or with anti-β-catenin antibody (rabbit polyclonal Ab, Upstate Biotechnology, #06-734), for 1 h. After extensive washing in PBS containing 0.1% Triton X-100, the samples were incubated with Alexa Flour 488 or Alexa Flour 594-conjugated goat anti-rabbit IgG (Invitrogen) for 1 h. The nuclei were stained with Hoechst 33258, and the mounted samples were scanned with an LSM 710 (Carl Zeiss).

To test the expression of nestin, insulin and c-peptide during the induction, cell clusters were sampled individually following each induction stage, respectively fixed in 4% paraformaldehyde for 20 to 30 minutes, triple-washed with PBS, and stained with mouse antibodies against human nestin (Abcam, Cambridge, Massachusetts, UK) and insulin (Invitrogen), and rabbit anti-human c-peptide antibody (Abcam), and then with secondary fluorescent antibodies including fluorescein isothiocyanate-conjugated donkey anti-mouse IgG and Alexa Flour 594-conjugated goat anti-rabbit IgG (Invitrogen). To investigate the expression rate of IPCs and other endocrine cells in cell clusters, the cell clusters from each time induction were respectively digested, subcultured for 24 hours in a lysine-coated 24-well plate before fixation, stained with mouse anti-human insulin and glucagon, rabbit anti-human SST and PP antibodies (Invitrogen), and the relevant IgGs conjugated with fluorescence. Ten nonoverlapping visual fields were randomly chosen from each staining sample to count stained and nonstained cells under microscopy, and they were photographed using an inverted microscope. The proportion of cells immunoreactive to particular antigens was quantified according to the stained cell number divided by the summation number of stained cells and nonstained cells (n = 10).