Ab108319

A RIPA lysis buffer (NCM, No.WB3100) and protease and phosphatase inhibitors were used to lyse rat hippocampal tissue. We measured the protein concentration of the extracts using the BCA protein analysis kit (Beyotime, No.P0010‐1). It was determined that an equal amount of protein (40 mg) was loaded onto the SDS–PAGE gel and then transferred to the PVDF membrane (Immobilon‐P Membrane, IPVH0010). Nonspecific protein binding was blocked with fast blocking solution (Beyotime, No. P0252‐100), followed by incubation with primary antibodies diluted in 1× TBS‐T for 8 h. Primary antibodies used include P‐ERK1/2 (diluted 1:5000, Proteintech, 28733‐1‐AP), ERK1/2 (diluted 1:1000, Proteintech, 11257‐1‐AP), P‐CREB (diluted 1:1000, Affinity, af3189), CREB (diluted 1:500, Abcam, ab32515), BDNF (diluted 1:5000, Abcam, ab108319), and β‐tubulin (diluted 1:5000, Proteintech, 10094‐1‐AP). After primary antibody incubation, the membrane was washed with 1× TBS‐T buffer and then incubated with HRP‐conjugated anti‐rabbit secondary antibody (diluted 1:5000, Proteintech, SA00001‐2) for 1 h. Following another wash, the membrane was visualized using an ECL chemiluminescence kit (NCM, P10100), and images were captured using the Tanon 5200 Multi imaging system (Tanon). Image data analysis was performed using ImageJ software (NIH).

Immunohistochemistry was carried out as previously reported with minor modifications (Aricioglu et al., 2019 (link)). Brain tissue was fixed with 4% paraformaldehyde for 24 h. Dehydrated, embedded in paraffin, and cut into slices of 5 µm thickness. After a series of operations, including deparaffinization, antigen repair and blocking, sections were incubated overnight with primary antibodies: anti-BDNF (Abcam, ab108319, 1:200), anti-P2X7 (Proteintech, 28207-1-AP, 1:50), anti-NLRP3 (Abcam, ab214185, 1:200) and anti-Cleaved caspase-1 (Thermo Fisher, PA5-99390, 1:200) at 4°C, followed by secondary antibodies incubation. Then, the slices were colored with diaminobenzidine (DAB). Finally, after dehydration and drying, sections were observed using a light microscope. The density values are analyzed by Image pro-plus software.

Total protein was isolated from the brain tissues and adjusted to the same final concentration using the protein extraction kit (Beyotime Institute of Biotechnology, Shanghai, China) and BCA Assay Kit (BCA Protein Assay Kit, Solarbio). SDS-PAGE was used to separate the samples, which were then transferred to NC (nitrocellulose filter) membranes. After being blocked with 2% BSA for 60 min, the NC membranes were incubated overnight with primary antibodies at 4 °C. The following day, after washing three times with PBST(phosphate buffered saline tween-20), the membranes were incubated with the secondary antibody (Goat Anti-Mouse IgG(H+L), Alkaline Phosphatase conjugate, and Goat Anti-Rabbit IgG(H+L), Alkaline Phosphatase-conjugated, Proteintech, China) for 1 h at room temperature. The membranes were washed four times in PBST buffer and incubated with a commercial western immunoblotting detection reagent (western blue stabilized substrate for alkaline phosphatase, Promega, USA) for 5 min. The resulting immuno-reactive bands were exposed by Amersham Imager 600 (GE). The primary antibodies used in this study were PKA (Abcam, ab108385,1:1000), CREB (Abcam, ab32515, 1:1000), pCREB (Abcam, ab32096, 1:1000), BDNF (Abcam, ab108319,1:1500), and GAPDH (Proteintech, 60004-1-Ig, 1:2000).