Nptx2

Sections of paraffin-embedded brain slices were deparaffinized and rehydrated before blocking non-specific antigen binding with 5% bovine serum albumin (Boster Biological Technology, China). The following primary antibodies were used: NPTX-1 (1:50; Abcam, Cambridge, UK), NPTX-2 (1:200; Abcam, Cambridge, UK), and CRP (1:100; Abcam, Cambridge, UK). Goat anti-rat/rabbit (Beijing Zhongshan Jinqiao Biotechnology, China) polymer enhancer and goat-resistant rabbit/rat IgG-conjugated polymer were added to each tissue section. Afterward, the immunostained sections were developed in diaminobenzidine (Beijing Zhongshan Jinqiao Biotechnology, China) and counterstained with a weak solution of hematoxylin solution. The stained tissue slices were visualized on a microscope (DMI6000B, Leica, USA).
The immunofluorescence staining protocol on the first day was the same as the immunohistochemical staining protocol, which was performed to evaluate the localization of NPTX-1, NPTX-2, and CRP. The luciferin antibody (100 µL) was added to each stained section. After incubating the sections in the dark at room temperature for 1 h, a drop of 4′,6-diamindino-2-phenylindole (Beijing Zhongshan Jinqiao Biotechnology, China) staining agent was added to each tissue section. An anti-quenching agent was added to seal the sections. The staining results were observed under a fluorescence microscope.

Immunohistochemical studies were performed using cryocut hippocampal sections (coronal, 16 µm) as previously described [28 (link)], with slight changes in blocking. Sections were blocked with either normal chicken serum (Nptx2, Vector S3000, Vector Laboratories, Burlingame, CA, USA) or normal goal serum (Npas4, Vector S1000, Vector Laboratories) for one hour at room temperature. Sections were incubated with different primary antibody concentrations in antibody solution (2.5% BSA in PBS) at 4 °C overnight (Nptx2, 1:100, Abcam, Cambridge, England; Npas4, 1:200, Abcam). Following primary antibody incubation, slides were washed in 1xPBS three times for five minutes each at room temperature. Slides were then incubated with appropriate fluorescent secondary antibody (1:500, Alexa-488 and 594, Invitrogen, Carlsbad, CA, USA) in antibody solution for 1.5 h at room temperature. The slides were then rinsed in 1× PBS three times for five minutes each and cover slipped using VectaMount (Vector Laboratories). Sections were viewed and images were captured using a Nikon Eclipse Ni microscope equipped with DS-Qi1 monochrome, cooled digital camera and NIS-AR 4.20 Elements imaging software. CEPO + IGF-1 and vehicle-treated sections were captured using identical exposure sections. Sections = −3.30 mm from Bregma.

Immunohistochemistry was performed using an immunohistochemistry kit (Maixin Biotechnology, Fuzhou, Fujian, PR China) according to the manufacturer’s instructions. Briefly, tissue samples were paraffin-embedded, cut into 4-μm sections, and incubated with primary antibodies against NPTX2 and Ki-67 (Abcam) at 4°C overnight. After DAB staining, the sections were imaged with an optical microscope (Olympus), and staining intensity was assessed according to the German immunohistochemical scoring system (15 (link)).