Quantitative Analysis of p21-Positive Cells in Dentate Gyrus

Frozen brains were serially sectioned in a coronal plane covering the entire length of the dentate gyrus (bregma point −2.3 to −6.3 mm) according to the coordinate Atlas [35 ] using a cryostat (Cryostat Series HM550 Microm international, A.S. Science Co., Ltd., Walldorf, Germany). p21 immunofluorescence staining was performed to investigate the cell cycle arrest as described previously [6 (link),21 (link)]. Forty micron sections were preserved in a cryoprotective buffer and stored in 24-well plates at 4 °C. Sections were incubated with an anti-p21 antibody (1:100, Santa Cruz Biotechnology, Texas, USA) at 4 °C overnight. Sections were incubated with an Alexa Fluor 488 rabbit anti-mouse IgG secondary antibody (1:300 Invitrogen, San Diego, CA, USA) at room temperature for 60 min and counterstained with propidium iodide (PI) (1:6000, Sigma-Aldrich, St. Louis, MO, USA). A systematic random sampling method was employed to choose every 8th section throughout the length of the dentate gyrus [36 (link)]. Nine sections were used for staining using a free floating method. p21-positive cells within three cells of the inner edge in the upper and lower blade of the DG were counted [10 (link),11 (link),37 (link)]. Sections were viewed at 10 × and 40 × by a Nikon ECLIPSE 80i fluorescence microscope (Melville, NY, USA).