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Title: Hippocampal neurons immunostained with MAP2 (green), BDNF (red) and Dapi (blue) at 20x magnification with scale bars, 20 μm.
Description: For immunostaining, primary hippocampal neurons were washed three times with 1X PBS, fixed in 4% paraformaldehyde for 10 min or with chilled methanol over night, washed again with 1X PBS and incubated first with monoclonal or polyclonal primary antibodies (Sheep anti BDNF;Abcam and Rabbit anti MAP2;EMD millipore) and then with the Cy2 or Cy5 conjugated secondary antibodies. Following secondary antibody incubation, coverslips containing hippocampal neurons were rinsed in 1X PBS, mounted on slides by Fluoromount (Sigma) and imaged using an Olympus BX41 fluorescent microscope equipped with a Hamamatsu ORCA-03G camera.