Vs asw oil 2

Free-floating 8–9 vibratome sections from each animal were washed 3 times for 10 min in Tris-buffered saline (TBS) (pH 7.4), immersed in endogenous peroxidase blocking solution for 30 min, and were incubated in preheated Target Retrieval solution at 85°C for 40 min (Dako, EnVision System HRP). Tissue sections were then incubated at 4°C overnight in a solution containing the rabbit anti-BDNF polyclonal antibody (diluted 1: 500) (AB1779, Merck Millipore). The next day, sections were washed 3 times for 10 min with buffer (1% BSA and 0.3% Triton-X in TBS) and incubated in buffer (1% BSA in TBS) added goat anti-rabbit IgG (1:200) for 2 h at room temperature. Sections were washed 3 times for 10 min in TBS and then visualized with 0.1% 3, 3’-diaminobenzidine containing 0.3% H₂O₂ in TBS for 7 min and washed by TBS 3 times for 10 min. Sections were then mounted on the gelatin-coated slides and dehydrated with alcohol gradient and cleared with xylene.
Images of whole immunostained sections were taken by the Virtual Slides System VS120 (Olympus) including the software VS ASW OIL 2.7 (Olympus Soft Imaging Solutions GmbH). ImageJ software was used to analyze the images of immunostained sections and calculated the mean optical density of BDNF-positive areas in subregions of hippocampus (Figure 2).

Free-floating 8 to 9 coronal sections per animal were washed 3 times for 10 minutes in Tris-buffered saline (TBS) (pH 7.4), immersed in endogenous peroxidase blocking solution for 30 minutes, and incubated in preheated Target Retrieval solution at 85°C for 40 minutes (Dako, EnVision System HRP). Tissue sections were incubated at 4°C overnight in a solution containing the rabbit anti-BDNF polyclonal antibody (diluted 1: 500) (AB1779, Merck Millipore). Then, sections were washed 3 times for 10 minutes with buffer (1% BSA and 0.3% Triton-X in TBS) and incubated in buffer (1% BSA in TBS) added goat anti-rabbit IgG (1:200) for 2 hours at room temperature. Finally, sections were washed 3 times for 10 minutes in TBS and then visualized with 0.1% 3, 3’-diaminobenzidine containing 0.3% H₂O₂ in TBS for 7 minutes and washed by TBS 3 times for 10 minutes. Sections were then mounted on the gelatin-coated slides and dehydrated with alcohol gradient and cleared with xylene.
Images of immunostained sections were taken using an Olympus BX61VS Scan microscope (objective: 10X; Olympus) equipped with a PIKE digital camera using the software VS ASW OIL 2.7 (Olympus Soft Imaging Solutions GmbH). ImageJ software was used to analyze the images of immunostained sections and calculated the mean optical density of BDNF-positive area in subregions (DG, CA1, and CA2/3) of hippocampus (Figure 3).