Cfx maestro 1.0 system

Total RNA from mouse brain tissue and BV2 was extracted using TRIzol (15596018, Ambion, USA) and quanti ed with a NanoDrop spectrophotometer(NanoDrop ONE C, Thermo Fisher Scienti c, USA). Then the cDNA synthesis was conducted using 5× PrimeScript RT Master Mix (TaKaRa) according to the manufacturer's instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using 2×QuantiNova SYBR Green (QIAGEN) to analyze gene expression on the Bio-Rad CFX Maestro 1.0 system. The relative mRNA expression was analyzed by using the 2-ΔΔCT method and normalized to GAPDH. Primer pair sequences are shown in Table 1.
Table 1. DNA sequences of primers used in PCRs and expected product sizes. The immunohistochemistry of brain sections was performed according to the DAB Detection Kit's instructions4 (GK600511, Gene Tech(shanghai, China). After returning to room temperature, the sections were washed with phosphate buffer saline(PBS), added endogenous peroxidase blocker and incubated for 20 minutes in the dark, then incubated with anti-PDCD4 (1:100, CST, USA) overnight at 4℃and added with horseradish peroxidase (HRP) at room temperature for 30 minutes, nally, the sections were stained with diaminobenzidine (DAB) and undergo gradient dehydration, drying and resin sealing, The stained sections were observed by microscope(ECLIPSE Ni-U, Nikon, Japan) on NIS-Elements F 4.6 system.

Total RNA from mouse brain tissue and BV2 was extracted using TRIzol (15596018, Ambion, USA) and quanti ed using NanoDrop spectrophotometer (NanoDrop ONE C, Thermo Fisher Scienti c, USA). Next, the cDNA synthesis was conducted using 5× PrimeScript RT Master Mix (TaKaRa) in line with the manufacturer's instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using 2×QuantiNova SYBR Green (QIAGEN) to analyze gene expression on the Bio-Rad CFX Maestro 1.0 system. The relative mRNA expression was analyzed by using the 2-ΔΔCT method, normalized to GAPDH. Primer pair sequences are shown in Table 1.
Table 1 DNA sequences of primers used in PCRs and expected product sizes. The immunohistochemistry of the brain sections was performed according to the DAB Detection Kit's instructions (GK600511, Gene Tech, Shanghai, China). After returning to room temperature, the sections were washed with phosphate-buffered saline (PBS). After blocking endogenous peroxidase for 20 min in the dark, the sections were incubated with anti-PDCD4 (1:100, CST, USA) overnight at 4°C. Next, they were incubated with horseradish peroxidase (HRP) at room temperature for 30 min and stained with diaminobenzidine (DAB), followed by gradient dehydration, drying, and resin sealing. The stained sections were observed using a microscope (ECLIPSE Ni-U, Nikon, Japan) on NIS-Elements F 4.6 system.