Cy3 conjugated affinipure donkey anti mouse igg

The slides were thawed and washed three times for 10 minutes with 0.01 mol/L PBS containing 0.1% Tween-20. Subsequently, the sections were incubated with blocking buffer containing 5% normal bovine serum and 0.3% Triton X-100 in 0.1 mol/L phosphate buffer for 1 hour at room temperature. The sections were then incubated with mouse anti-rat glial fibrillary acidic protein monoclonal antibody (1:6,000, Calbiochem, Darmstadt, Germany) or mouse anti-rat synaptophysin monoclonal antibody (1:4,000, Sigma-Aldrich, St. Louis, MO, USA) overnight at 4°C. After washing, the sections were incubated with Cy™ 3-conjugated AffiniPure donkey anti-mouse IgG (1:400; Jackson Immuno Research, West Grove, PA, USA) for 2 hours at room temperature. Cell nuclei were stained for 8 minutes with Hoechst 33258 staining solution (Beyotime Institute of Biotechnology, Haimen, Jiangsu Province, China). Finally, the sections were mounted in 1:1 0.1 mol/L phosphate buffer and glycerol (by volume) and stored at 4°C in the dark. Negative controls performed with the secondary antibody alone showed no staining. All images were captured using a laser scanning fluorescence microscope (Nikon 80I, Tokyo, Japan).