The brains were removed from the RNAlater buffer and the full brain was homogenized in 1 ml QIAzol Lysis Reagent per 100 mg tissue (Qiagen, supplied with kit) using a TissueLyser (Qiagen cat no: 85220).
Wt and hyh mice (Table 1) were anesthetized and transcardially perfused with 2.5 % glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, and the brains were dissected out and immersed in fresh fixative for 1 h.
The brains were removed and placed in 4% buffered formaldehyde overnight at 4°C, and the brains were then cryoprotected by overnight immersion in 30% sucrose.
Briefly, after perfusion with 0.9% saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer, the brains were post-fixed, cryoprotected with 30% sucrose until they sank, and frozen sectioned (20 µm) in the coronal plane of the dorsal hippocampus (∼2.5 4.5 mm posterior from bregma).
After rinsing in 0.1 M phosphate buffer (pH 7.4), the brains were sectioned on a vibratome (Leica VT1000S) in the coronal plane at a thickness of 200 μm.
Next day, hemispheres were stored in 20% glycerol, 2% DMSO in phosphate buffer (PB) until the brains were sliced as consecutive serial coronal sections (5 series, 50 μm thickness).
After transcardial in vivo perfusion (0.4% paraformaldehyde pH 7.4, in 0.1 M phosphate buffer solution), the brain was quickly removed and placed in 30% sucrose solution in phosphate buffer.
It is critical to remember to scale back the obtained concentrations (or areas under the concentration-time peaks) to the undiluted buffer and brain slice concentrations by multiplying by the dilution factors as appropriate.
The CSF macrocirculation functions as a closed hydraulic system, which provides a mechanical buffer between the skull and the brain, and relieves the effects of gravitational and accelerational stresses [ 21].
Briefly, mice were transcardially perfused with 4% paraformaldehyde in 100-mM phosphate buffer, and brains were dissected and preserved for 24 h in the same buffer.
