A temporal single-nucleus atlas of the hippocampus in temporal lobe epilepsy mice unveils neuroprotective IGF1+ microglia
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The original western blot images of this article.
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Nuclear and cytoplasmic proteins were extracted following the instructions provided with the kit (EX1470; Solarbio; Beijing, China). Cells were lysed using radio immunoprecipitation assay lysis buffer (RIPA) lysis buffer (P0013B; Beyotime) containing protease and phosphatase inhibitors. The lysates were centrifuged at 16,000 × g (4 °C, 30 min), and the supernatant was collected. Protein was quantified using a BCA protein assay kit (P0010; Beyotime). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membrane (Millipore; USA). The membranes were blocked with a rapid protein-free blocking buffer (PS108P; Epizyme; Shanghai, China) at room temperature for 30 minutes and incubated with primary antibodies at 4°C overnight. After 12-18 hours, membranes were washed with tris-buffered saline containing 0.1% tween-20 (TBST) and subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 hour. Membranes were imaged using enhanced chemiluminescence reagent (BL520B; Lanjieke; Beijing, China) on a Fusion imaging system (Vilber Lourmat, Marne-la-Vallée, France) and quantified using ImageJ software.
Institutions
- Chongqing Medical University First Affiliated HospitalSichuan, Chongqing