Anti rabbit cy2 secondary antibody

Cells were fixed in 4% paraformaldehyde (PFA) for 20 minutes at room temperature and then permeabilized using blocking solution (10% (v/v) horse serum in PBS with 0.1% Tween 20 (0.1% PBST)) for 60 minutes at room temperature. Cells were then incubated with the anti-Nestin antibody (Abcam, Cambridge, England, Ab5968; 1/800 in blocking solution) overnight at 4°C. After washing in 0.1% PBST, cells were incubated with the anti-rabbit Cy2 secondary antibody (Jackson, Pennsylvania, USA, 711-225-152; 1/200 in blocking solution) for two hours at room temperature. After washing in 0.1% PBST, cells were then incubated in 300 nM 4′,6-diamidino-2-phenylindole (DAPI) for 10 minutes at room temperature after which they were washed twice in 0.1% PBST and viewed under fluorescence microscopy. For quantification of Nestin expression, ImageJ was used to calculate the Nestin:DAPI ratio. For each field, the overall fluorescence intensity was calculated for each color channel and from this the ratio of Nestin:DAPI fluorescence was determined.