Z0334

Vibratome sections (Leica VT1000S) (30 μm) were washed 3 times in PBS, blocked in 10% normal goat serum, 1% bovine serum albumin, 100 mM glycine, 0.1% Triton-X, in PBS for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C in 1% normal goat serum, 1% bovine serum albumin, 0.1% Triton-X, in PBS. Samples were then washed in PBS and incubated in secondary antibodies for 1 h at room temperature. Primary antibodies used were: guinea pig anti-DCX (1:2000, Millipore, AB2253), rabbit anti-GFAP (1:1000, Daco, Z0334), rat anti-BrdU (1:1000, Bio-Rad, OBT0030G), mouse anti-L1cam (1:300, Abcam, [2C2] ab24345), rabbit anti-Sox2, (Millipore, ab5603) (1:2000), mouse anti-PCNA (Abcam, ab29) (1:500). PCNA was detected with antigen retrieval in sodium citrate buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) at 95 °C hot plate for 45 min, cooled down for 20 min, and washed 2x in PBS for 5 min. Secondary antibodies used were goat anti-mouse, -rabbit, -guinea pig, and -rat Alexa Fluor 488, 568, and 647. For BrdU staining, before the blocking step, sections were incubated in 2 M HCl at 37 °C for 30 min, followed by 0.1 M sodium borate, pH 8.5 for 20 min, and PBS. Sections were imaged using a Zeiss AxioSkop2 or a confocal Zeiss LSM880 Airyscan Confocal Microscope.