Gap 43

Brain tissues were lysed with RIPA buffer containing a cocktail of protease inhibitors and phosphatase inhibitors. Lysate was centrifuged at 14,000 rpm at 4 °C for 15 min and the supernatant was collected. The total protein concentration was determined by using the Bradford assay (Bio-rad Laboratories, Hercules, CA, USA). Equal amounts of protein were loaded on 10% SDS-PAGE gel and then transferred onto a PVDF membrane (Millipore, Temecula, CA, USA). After blocking with 5% skim milk or 3% BSA, the membrane was incubated overnight at 4 °C with primary antibodies against BDNF, TrkB, PSD-95, Erk1/2, pErk1/2 (Abcam, Cambridge, UK), and GAP-43 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing with TBS-T, the membrane was incubated with the proper secondary antibody conjugated to HRP for 2 h at room temperature. Interested protein signals on the membrane were detected by using SuperSignal West Pico chemiluminescence substrate (Thermo Fisher Scientific, Rockford, IL, USA). The signal intensity of each band on the film was quantified by using ImageJ software (Java 8) and normalized to the corresponding β-actin (Cell Signaling Technology, Danvers, MA, USA) as an internal control.

A western blot analysis was used to measure GAP‐43, GFAP, c‐fos, ERK, and p‐ERK expression in the retinas of the Bifidobacterium‐treated mice. The retinas were collected and lysed. An equal amount of protein was added to 10% SDS‐PAGE gels, electrophoresed, and transferred electrophoretically onto polyvinylidene difluoride membranes. The membrane was blocked with a blocking buffer (Tris‐buffered saline Tween‐20 (TBST), containing 5% nonfat dry milk) for 1 h at room temperature and then incubated overnight with primary antibodies directed against GAP‐43 (Santa Cruz Biotechnology, 1:100), GFAP (Abcam, 1:1000), c‐fos (Abcam, 1:1000), ERK (CST, 1:1000), p‐ERK (CST, 1:1000), and GAPDH (Proteintech, 1:1000). The membranes were washed with TBST for 30 min and then incubated with a secondary antibody (Proteintech, 1:5000) for 1 h at room temperature. Proteins were visualized using a molecular imaging system (Amersham Imager 600, GE Healthcare, Buckinghamshire, UK). The experiments were repeated three times.

The dried powder of herbal medicine Semen Persicae extract was purchased from a local Pharmaceutical company Nong's Company (Hong Kong). Antibodies against ERK1/2, phospho-ERK1/2, β3-tubulin, goat anti-rabbit IgG secondary antibody, and Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody were obtained from Cell Signaling Technology (Boston, MA, USA). Monoclonal antibodies against growth associated protein-43 (GAP-43), microtubule associated protein 2 (MAP2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fluorescein isothiocyanate conjugated phalloidin (FITC-phalloidin) and other chemicals were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA) unless indicated otherwise.