Horseradish peroxidase conjugated goat anti rabbit igg secondary antibody

Hippocampal tissues were homogenized in RIPA cell lysate (Beyotime, P0013B), centrifuged at 12,000 × g for 15 min, and the supernatant was collected. Each protein sample was loaded into the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel spiked wells. Electrophoresis was performed at constant pressure of 80 V for approximately 1 h. The proteins were transferred to a polyvinylidene fluoride membrane (Millipore, IPVH00010), fixed with western closure solution (5% skim milk powder), slowly shaken on a shaker for 2 h at room temperature, and then incubated with the following primary antibodies: rabbit anti-postsynaptic density-95 (PSD-95) antibody (1:2000, abcam, ab238135), rabbit anti-synaptophysin (SYN) antibody (1:1000, Bioss, bs-8845R), rabbit anti-BDNF antibody (1:1000, abcam, ab108319), and rabbit tyrosine kinase receptor B (TrkB) antibody (1:5000, abcam, ab187041). The samples were further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:20000, Zsbio, ZB-2301) for 1.2 h. The membranes were washed with phosphate-buffered saline with Tween and the ECL luminescence kit (Thermo, 340,958) was used to detect the proteins. Finally, the intensity of the bands was analyzed by Image J software (Media Cybernetics, United States).

Briefly, 4 μm thick paraffin-embedded tissue sections were deparaffinized, rehydrated, treated with 0.3% hydrogen peroxide, and subjected to antigen retrieval with heat induction for approximately 10 min. The following primary were used for IHC staining: rabbit anti-CD66b (1:200, Ab197678, UK) and rabbit anti-CD31 (1:200, Ab76533, UK). The tissues were then incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (ZSGB-bio, Beijing, China). The slides were finally stained with 3,3´-diaminobenzidine (DAB substrate kit, ZSGB-Bio, Beijing, China) and counterstained with haematoxylin. The specimens were analysed under a light microscope (Nikon, Tokyo, Japan) by pathologists.