Alexa fluor 647 conjugated goat anti rabbit secondary antibody

After the sacrificed animals were perfused with 4°C saline and 4°C paraformaldehyde (PFA), the brains were removed and placed in 4°C paraformaldehyde for 24 h. After 15% and 30% sucrose gradient dehydration, the brain tissues were stored at -70°C until they completely sank to the bottom. A cryostat microtome (Microm HM 550, Thermo Fisher Scientific Inc., Germany) was used to cut 20 μm thick coronal brain slices for immunofluorescence. Slices containing the hippocampal CA1, CA3, and DG region (−2.3 mm to −4.16 mm from the bregma) were chosen by the experimenters. Brain slices were blocked with 10% normal goat serum solution at 37°C for 2 h and then incubated for 18−20 h at 4°C with an anti-MasR antibody (rabbit polyclonal, 1 : 500, Alomone, Israel) or an anti-AT1R antibody (rabbit polyclonal, 1 : 500, Alomone, Israel). After washing with PBS, the slices were incubated with an Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody (1 : 1000, Jackson ImmunoResearch Laboratories, USA) at 37°C for 1.5 h. Finally, the slices were visualized using a Nikon Eclipse Ti confocal microscope (Nikon, Japan) and images in the hippocampal CA1, CA3, and DG region were captured using NIS-Elements software (Nikon, Japan).