Primary antibodies against bdnf

The expression of BDNF protein in the hippocampus was evaluated by Western blot analysis. The concentrations of proteins extracted with TRizol reagent (Thermo Fisher Scientific, Inc., Tokyo, Japan) were determined using Bradford’s method with bovine serum albumin (BSA) as a standard. Each amount of proteins was subjected to SDS-PAGE and then transferred to polyvinylidene difluoride membranes (0.45 µm, Merck Millipore, Billerica, MA, USA). The membranes were blocked with 3% BSA in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBST) for 1 h at room temperature, and incubated overnight at 4 °C with primary antibodies against BDNF (1:3000; Abcam, Cambridge, MA, USA) and tubulin (1:5000; Abcam). Next, the membrane was washed with TBST three times and incubated with secondary horseradish peroxidase-conjugated antibody (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 1 h. The protein bands were visualized using the EzWestLumi plus kit (ATTO, Tokyo, Japan) and AE-9300 Ez-Capture (ATTO). The intensity of protein bands was analyzed using ImageJ software (NIH Image, Bethesda, MD, USA).

Sliced sections were air dried and incubated in frozen methanol (2 min) and in 4 % Para-formal-aldehyde (4 min). After three washes in phosphate-buffered saline (PBS) containing Tween 20 (PBS/T) (Sigma-Aldrich), the sections were incubated for 60 min in a blocking solution in normal goat or horse serum in PBS and then overnight at 4 °C with the primary antibodies against BDNF (1:250 each; Abcam). After three washes in PBS/T, sections were incubated in DyLight-488 in PBS containing 2 % normal serum for 2 h. Sections were washed and mounted with mounting medium (Vectrastain Vector laboratories, USA). Control staining was performed in the absence of the primary antibodies. Additionally, secondary fluorescent labels were swapped to check cross-reactivity and sections were incubated without any primary antibodies to check for any non-specific binding of the secondary antibodies.