C reactive protein (crp)

In CRP and SAA treatment studies, THP-1 or HUVEC cells were cultured to the control, i.e., 0.1% bovine serum albumin (BSA) in phosphate buffer saline (PBS), or CRP (Abcam, 1 μg/mL) or SAA (SantaCruz, 0–1 μg/mL) or both of them for 2–6 h. At the end of the incubation, cell pellets were collected for RNA isolation while the supernatants were for the quantification of chemokines by ELISA.

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.