Rabbit anti apkc λ

The tissue sections were incubated at room temperature with 3% H₂O₂ solution for 20 min to block the endogenous peroxidase activity. After being washed three times with PBS, the sections were pre-incubated in PBS containing 0.1%Triton X-100 and 5% normal goat-serum for 30 min, then incubated again overnight with the following primary antibodies at 4°C: rabbit anti-aPKC-λ (1:50, Santa cruz), rabbit anti-PAR3(1:300, Abcam), rabbit anti-LGL1(1:40, Santa Cruz), mouse anti- NeuN(1:1,500, Chemicon). After being washed by PBS, the sections were re-incubated with biotin-labeled anti-rabbit or anti-mouse immunoglobulin (Ig) G secondary antibodies which were raised in a goat (1:200, Cwbiotech) at room temperature for 2 h. Afterwards, these were washed in PBS and incubated in avidin-biotin-horseradish peroxidase complex (1:200, Vector laboratories) at room temperature for another 2 h. Finally, the sections were processed with 3,3′-diaminobenzidine tetrahydrochloride solution (Zsbio) for visualization. Following this, the slides were routinely washed, dehydrated, and mounted. Mean optical density (OD) was calculated by Image pro plus 6. The total number of NeuN(+) cells in CA3 and hilar region of five sections, which were randomly selected from every rat, were calculated at 40× magnification of the microscope.

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.