Hrp labeled goat antirabbit igg secondary antibodies

The proteins were extracted from hippocampal tissues using RIPA lysate buffer (Beyotime, China), and then concentrations of proteins were measured using bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, China). Proteins were separated by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes, which had been washed by using Tris Buffered Saline Tween (TBST), and then were blocked with 5% skimmed milk in TBST (room temperature, 2 h), The membranes were incubated at 4°C overnight with primary antibodies. The primary antibodies included rabbit anti-NDR2 (1:3000, Affinity, USA), and rabbit anti-GAPDH (1:10,00, Servicebio, China). Unbound antibodies were washed by TBST, then the membranes were subsequently incubated with HRP-labeled goat antirabbit IgG secondary antibodies (1:200, Beyotime, China) for 1 h. The immunoreactive bands were visualized by using a chemiluminescence (ECL) kit (Beyotime, China), Images were acquired with a Chemiluminescent Gel Imaging System (FluorChem FC3, ProteinSimple, USA), and densitometric analysis was performed with ImageJ software.

Proteins were extracted from hippocampal tissues using RIPA lysate buffer (Beyotime), and then concentrations of proteins were measured using bicinchoninic acid (BCA) Protein Assay Kit (Beyotime). Proteins were separated by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. Membranes. which had been washed by using Tris Buffered Saline Tween (TBST) and had been blocked with 5% skimmed milk in TBST (room temperature, 2 h), were incubated at 4°C overnight with primary antibodies. The primary antibodies included rabbit anti-aPKC-λ (1:1,000, Santa Cruz), rabbit anti-PAR3 (1:1,000, Abcam), rabbit anti-LGL1 (1:100, Santa Cruz), and rabbit anti-GAPDH (1:10,000, Proteintech). Unbound antibodies were washed by TBST, then the membranes were subsequently incubated with HRP-labeled goat antirabbit IgG secondary antibodies (1:200, Beyotime) for 1 h. The immunoreactive bands were visualized by using chemiluminescence (ECL) kit (Beyotime), and the optical density (OD) of these bands were quantified by using the Image J software.

At different time points, rats in the control and PTZ groups were deeply anesthetized with chloral hydrate and immediately decapitated for western blot analysis. Tissues were snap-frozen in liquid nitrogen, and proteins isolated from the hippocampus were extracted using RIPA lysate buffer (Beyotime Co.). All proteins were denatured (95°C, 10 min) and chilled on ice (5 min), then electrophoresis was performed (10% SDS-PAGE). The proteins were transferred onto 0.45- or 0.22-μm polyvinylidene difluoride membranes (Pall Corporation, Port Washington, NY, USA), washed three times with TBST, and then blocked with 5% skimmed milk in TBS (room temperature, 2 h). The membranes were incubated at 4°C overnight with the appropriate primary antibodies (anti-RGMa rabbit anti-rat polyclonal antibody, 1:800; anti-FAK Tyr397 rabbit anti-rat polyclonal antibody, 1:500; anti-Ras rabbit anti-rat polyclonal antibody, 1:500; anti-GAPDH rabbit anti-rat polyclonal antibody, 1:1,000). Unbound antibodies were washed, and the membranes were subsequently incubated with HRP-labeled goat anti-rabbit IgG secondary antibodies (1:2,000 dilutions; Beyotime Co.) for 1 h (room temperature). After washing (3×15 min), the immunoreactive bands were visualized by enhanced chemiluminescence (Bio-Rad imaging system; Bio-Rad, Hercules, CA, USA) and quantified using Image Lab software (Bio-Rad).