The experimental mice were anesthetized by intraperitoneal injection of 4% chloral hydrate and then transcardially perfused with saline followed by 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Mouse brains were dissected, post-fixed for 4–6 h in 4% PFA at 4 °C, and subsequently cryo-protected in 20% sucrose in PBS for 12 h followed by 30% sucrose for 24 h. Tissue blocks were prepared by embedding in Tissue-Tek O.C.T. Compound (Sakura 4583). The brain sections (20 μm in thickness) were prepared using a cryostat microtome (Leica, CM3050S), dried for 30 min at room temperature in the dark and stored in −20 °C freezer. For immunostaining, the tissue sections were washed with 1×TBS (pH = 7.4, containing 3 mM KCl, 25 mM Trizma base, and 137 mM NaCl) and pre-blocked with 1×TBS + + (TBS containing 5% donkey serum and 0.3% Triton X-100) for 1 h at room temperature, followed by incubation with primary antibodies diluted in TBS + + overnight at 4 °C. The primary antibodies used in this study included Sox2 (Goat; R&D Systems; #KOY0317071; 1:500), NG2 (Rabbit; Millipore; #3018740; 1:200), Olig2 (Goat; R&D Systems; #UPA0617051; 1:200), NeuN (Mouse; Abcam; #ab104224; 1:1000), HuC/D (Mouse; Molecular Probes; #A21271; 1:1000), CC1 (Mouse; Calbiochem; #OP80; 1:200), S100 (Rabbit; Abcam; #ab868; 1:400), Iba1 (Rabbit; Wako; #019-19741; 1:1000), GFP (Goat; Rockland; #33302; 1:1000), RFP (Rabbit; Rockland; #35055; 1:1000), GFAP (Rabbit; DAKO; #Z0334; 1:1000), Igf1r (Rabbit; CST; #30275; 1:200), Sox9 (Rabbit; Abcam; #ab185966, 1:250);PDGFRα (Goat; R&D Systems; #AF1062; 1:200), Vimentin (Rabbit; Abcam; #ab92547; 1:500), Mia (Goat; R&D; #AF2050, 1:50), Ki67 (Rabbit; Abcam; #ab16667;1:500) and mCherry (Rat; ThermoFish; #SI259077; 1:500). After primary antibody incubation, the brain sections were washed for three times with 1×TBS and incubated with following secondary antibodies for 2 h at room temperature: anti-rabbit/mouse Cy2, anti-goat/rabbit Cy3 and anti-rabbit/goat Cy5 (Donkey; Jackson ImmunoResearch; 1:500).

For EdU labeling, the tissue sections were permeabilized with 0.5% Triton-X-100 for 30 min and detected with detection solution containing 5 µM Sulfo-Cy3/Cy5 azide (Lumiprobe, #C1330/#C3330), 0.1 M Tris-HCl (pH = 7.5), 4 mM copper sulfate, and 100 mM sodium ascorbate for 30 min. After staining, sections were coverslipped with mounting medium, air-dried overnight and maintained at 4 °C in the dark for further imaging. The brain sections were imaged using Zeiss LSM 710 confocal microscopy (Carl Zeiss, Oberkochen, Germany) and Leica SP8 confocal (Leica, Germany) equipped with four lasers (405, 488, 568, and 647 nm). Images were processed and analyzed using Image J software (NIH, Bethesda, MD, USA). For 3D reconstruction, optical stacks from the entire ME were serially aligned along the rostrocaudal axis using Reconstruct 1.1.0 (J.C. Fiala, NIH), followed by import into Imaris 9.0.1 (Bitplane) for further analysis.

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