Ab138064

Primary neurons with various treatments were fixed in 4% paraformaldehyde for 20 min. Followed by rinsing with phosphate-buffered saline (PBS). The cells were then blocked with 40 μl of blocking solution (10 μl sheep serum, 190 μl PBS, 0.6 μl Triton-100), followed by incubation with 30 μl of β-tubulin (TUJ1) and NEUN primary antibody or Navβ2 antibody (1 : 800, Abcam, Ab138064, UK) diluted with 2% sheep serum (1 : 1000, Abcam, Ab177487, UK) overnight at 4°C. Subsequently, the cells were stained with a secondary antibody at 37°C for 1 h. Finally, the sections were stained with 4′,6-diamidino-2-phenylindole (DAPI), which contained antifluorescence quenching agent (Biyuntian Co., Ltd., C1006, China), and observed and photographed under a fluorescence microscope (NIKON T1-SM, Ti-E, Japan).

Adherent primary neurons were collected using radioimmunoprecipitation assay buffer, whereas the tissue homogenates of cortical or hippocampal tissues of mice were dissolved in protein lysate (Biyuntian Co., Ltd., P0013C, China). Centrifugation was performed (12000 rpm, 15 min, 4°C), and the supernatant was absorbed as extracted protein.
In all, 40 μg of protein was extracted from each sample and isolated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein was then transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was placed in a blocking solution to reduce nonspecific binding. Subsequently, the membrane was washed with Tris-buffered saline Tween-20 (TBST).
Following this, the membrane was incubated with GAPDH antibody (1 : 1000, Abcam, Ab9485, UK), APP antibody (1 : 1000, Abcam, Ab241592, UK), Navβ2 antibody (1 : 1000, Abcam, Ab138064, UK), BDNF antibody (1 : 1000, Abcam, Ab108319, UK), or NEP antibody (1 : 1000, R&D Systems, AF1182, USA) at 4°C overnight. After washing with 1x TBST, the membrane was incubated with secondary antibodies (1 : 1000, Abbkine, A23220/A23410, USA) at room temperature for 1 h and rinsed with 1x TBST again. All results were photographed using Bio-Rad Gel Imaging System (ChemiDoc™ XRS+). ImageJ (National Institutes of Health, USA) was used for strip quantitative analysis.