Adult mice were anesthetized with sodium pentobarbital, and their cerebellum was surgically exposed. As depicted in Fig. 32.1a, while the heart was normally beating, the cerebellum was vertically cut with the precooled cryoknife as fast as possible and simultaneously poured with the isopentane –propane cryogen [11]. The frozen cerebellar tissues were carefully trimmed out with a dental drill in liquid nitrogen and then processed for the following FS step.

Under normal condition, IgG was immunolocalized in blood vessel s of cerebellum with IVCT followed by FS (Fig. 32.1c). Judging from sizes of tiny ice crystal s at low resolution by light microscopy , artificial destruction of blood vessel structures was not well recognized in molecular layers at cerebellar surface layer tissues (ML in Fig. 32.1c) and granular layers at deeper regions (GL in Fig. 32.1c). The well-preserved tissues were nearly within 200–300 μm from the cerebellar surface area, which the isopentane –propane cryogen was directly poured over and the cryoknife was closely attached to. There was no immunoreactivity for IgG outside the blood vessels, suggesting no leakage or prominent movement of serum IgG proteins during the FS method as well as the immunohistochemical staining process.

After the perfusion fixation with 2 % paraformaldehyde in phosphate buffer, there was no immunoreactivity with anti-IgG antibody in the blood vessel s , due to washing out of blood during the perfusion-fixation . Thus, soluble serum protein s , such as IgG, can be preserved by the combination of our IVCT and FS method.

In the cerebellar tissues immediately after a sudden stop of cerebellar blood supply, IgG was diffusely immunostained around blood vessel s (Fig. 32.1d). In such extravascular regions, there was no blood cell, indicating that no bleeding nor inflammatory reactions had happened around there. The distribution pattern of the IgG-immunostaining products was sometimes fan-shaped, indicating the leakage of serum IgG from some restricted points of the blood vessels. These findings suggest that the quick-freezing and freeze-substitution method applied in this section for immunohistochemistry enabled us to detect the functional damage of the BBB.