Cy3 conjugated secondary antibody

Coronal cryosections (10 μm) were obtained at the center of the lesion (Bregma −1 to +1 mm) from frozen brains of SAHA-treated, MC1568-treated and vehicle-control rats that were sacrificed 7 days after MCAO. Sections were mounted on polyethylene naphthalate (PEN) membrane slides (Leica Microsystems). To identify neurons, frozen sections were stained with a neuronal specific anti-NeuN antibody using a fast immunostaining protocol, as previously described (Liu et al., 2009a (link)). Briefly, frozen brain coronal sections were air-dried for 30s at room temperature and then immersed in ice-cold acetone for 2 min of fixation. After a brief rinse with 0.1% diethylpyrocarbonate-treated phosphate buffered saline (DEPC-PBS), sections were stained with a mouse anti-NeuN antibody (Millepore,1:50 dilution) for 5 min followed by a Cy3-conjugated secondary antibody (DAKO, 1:50 dilution) for 5 min. After a brief rinse with DEPC-PBS, sections were air-dried under laminar flow for 10 min and immediately used for LCM. Approximately 2,000 NeuN positive neurons were quickly collected from the peri-infarct cortex of each animal using a Leica LMD6000 Microsystem (Figure 4A).