Goat anti rabbit igg h l cy3 secondary antibody

After anesthesia, mice were immediately perfused transcardially with 4% paraformaldehyde (PFA). The brains were fixed in 4% PFA for 24 h after perfusion, and then soaked in 15%, 25% and 30% sucrose for 24 h, 24 h and 48 h. Next, they were immerged in OCT compound and frozen at -80°C. After rinsing with PBST for three times, sections were incubated in blocking solution containing 0.2% Triton X and 5% goat serum for 40 min. Then they were incubated with primary antibody for the detection of PSD95 (1:200, Abcam), Synapsin-1 (1:200, Abcam), and p-Synapsin (1:200, Arigo) at 4°C overnight. Rabbit IgG monoclonal Isotype Control (1:200, Abcam) served as negative control. On the second day, sections were incubated in Goat Anti- Rabbit IgG (H&L) Cy3 secondary antibody (1:500; Abcam) for 2 h, followed by incubation with DAPI (1 μg/mL; Boster Bio, Wuhan, China) for 5 min. Images were captured using a fluorescence microscope (BX51, Olympus, Japan) and the immunopositive cells was analyzed by ImageJ software (version 1.52, NIH, USA) to calculate the calibrated total fluorescence with this formula: Integrated density - (Area × Mean background fluorescence), and then normalized to achieve fold changes (% of control).