Publication protocol
A total of 6‐8 slides (spaced at 200 μm), on which each brain section contained intact hippocampal formation including CA1, CA3, and DG areas, were used for IHC and FIHC on BrdU, DCX, or GFAP. Sagittal brain sections were deparaffinized, ethanol rehydrated. After retrieval of antigen (boiled in 0.01M pH6.0 sodium citrate buffer solution for 25 minutes and then cooled to room temperature), sections were blocked using hydrogen peroxide (30% H2O2 diluted 10:1 in methanol for 30 minutes), incubated with BSA (5% bovine serum albumin in PBS for 30 minutes) and were incubated overnight at 4°C with the following antibodies including GFAP (1:500; Sigma‐Aldrich, St. Louis, MO, USA), BrdU (1:200; Abcam, Cambridge, UK), DCX (1:200; Santa Cruz Biotechnology, Santa Cruz, Dallas, TX, USA), NeuN (1:500; Millipore, Billerica, MA, USA), MAP2 (1:200; Sigma‐Aldrich), or SVP38 (1:500; Sigma‐Aldrich), respectively. After incubation with secondary antibodies diluted in PBS (phosphate buffer solution), the sections were developed using the avidin‐biotin peroxidase complex method (ABC kit). Sections were then dehydrated using graded ethanol and sealed using neutral resin. For fluorescence immunostaining, brain sections were incubated with either Alexa Fluor 488 goat anti‐mouse/anti‐rabbit or Alexa Fluor 594 goat anti‐mouse/anti‐rabbit secondary antibodies (Invitrogen, Waltham, MA, USA), and then analyzed with a Leica SP5 confocal laser‐scanning microscope.
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