Rabbit anti synaptophysin

Mouse brains were dissected and immediately fixed in 4% paraformaldehyde (PFA) (Sigma-Aldrich) for 24 hours at 4 °C. The frozen tissue sections were prepared as previously described [32 ]. The slices were blocked with blocking buffer (5% goat or donkey serum, 0.3% Triton X-100 in PBS, pH 7.4) for 1 hour, then incubated with primary antibodies at room temperature overnight. The following primary antibodies were used: mouse anti-PSD95 antibody (Cell Signaling Technology, 36233, 1:400), rabbit anti-Synaptophysin (Cell Signaling Technology, 5461, 1:500), rabbit anti-Aβ (Cell Signaling Technology, 8243, 1: 1,000). After washing with PBS, the slices were then probed with appropriate cross-adsorbed secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 594 (Thermo Fisher Scientific, 1:500). Images were collected under a Nikon confocal microscope followed by three-dimensional reconstruction and analysis using Nikon-Elements advanced Research software. Aβ-plaque occupied area were analyzed using “Threshold” operation to choose plaque then divided by the area of the brain. The synapses were defined by colocalization of synaptophysin and PSD95 which was analyzed by using “AND” operation in “binary operation” dialog of NIS element software to overlap two binary layers. The number of synapses was counted and divided by total volume of the image stack.