Immunofluorescence was performed to determine the changes of neurites and nuclei as previously described [10 (link),16 (link)]. Forty-eight hours after OGD injury, fixed in 4% paraformaldehyde (pH 7.4), cells were incubated with primary antibodies at 4°C overnight (rabbit anti-GAP-43 monoclonal antibody, 1:200, Cell Signaling Technology; rabbit anti-activated-caspase-3, 1:200, Beyotime; mouse anti-class III-β-Tubulin monoclonal antibody 1:200, Beyotime). After washing, they were further incubated with corresponding secondary antibodies (Cy3 donkey anti-mouse IgG, 1:400, Jackson Immunoresearch; Dylight488 donkey anti-rabbit IgG, 1:400, Jackson Immunoresearch). Nuclei were stained with Hoechst33342 (Sigma, USA). Glass slides were detected with a ZEISS LSM 710 confocal microscope (Carl Zeiss, Germany), and the length of axon and the number of dendrites were analyzed with LSM Image Browser (V4.2.0.121). Meanwhile, 24 hours after OGD injury, the number of apoptotic cells was calculated. For each group and experiment, 3 visual fields in every coverslip were observed and all trials were repeated three times.
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