Fig. 41.1
(a–c) Fluorescence micrographs of quick-frozen, freeze-substituted, and freeze-dried TRITC- and FITC-conjugated goat immunoglobulins, which were mixed on glass slide from different direction (arrows in a, b). (c) is the merged image of (a) and (b). (d) Schematic representation of the in vivo cryotechnique with fluorescent probe injection. (e, f) DIC (e) and fluorescence (f, Fluo) images of IVCT mouse liver where FITC-conjugated goat immunoglobulin was injected. Precise data have been reported in the previous paper (Terada et al. [8]). Bars: 100 μm
After the freeze substitution, the samples were treated with sucrose and then frozen with isopentane precooled with dry ice, and 6–8-μm sections were cut at −30 °C in a cryostat apparatus. After attaching the thawed tissues to the poly-L-lysine-coated glass slides, they were briefly washed in phosphate-buffered saline (PBS) to remove the sucrose. Then, the tissues on glass slides were covered using coverslips and observed under a fluorescence microscope.
41.3 Blood Circulation Is Visualized by Injection of Fluorescent IgG Probe with IVCT
Some well-preserved tissues without obvious ice-crystal formation at a light microscopic level were obtained within 300–400 μm of the frozen tissue surface, as revealed by differential interference contrast (DIC) (Fig. 41.1e) and DAPI staining. Such well-preserved areas were widely obtained by sectioning the frozen surfaces vertically.
Regarding the blood vessel s , including the hepatic sinusoid s of mouse livers, the injected fluorescent probe was retained after performing the in vivo cryotechnique and the freeze-substitution procedure (Fig. 41.1f). Red blood cells were strictly localized in various types of blood vessels, indicating that normal blood flow was retained even after the injection of goat FITC-IgG , when the in vivo cryotechnique was performed. The paraformaldehyde fixation during the freeze substitution did not alter the fluorescent characteristics of the fluorescent probe. Its light emission occurred at the same wavelength as that of the probe in water only. Some interlobular veins (ILV in Fig. 41.1e, f) surrounded a segment of the hepatic lobul e. Many venous capillaries at the surface of the hepatic lobule arise from interlobular veins and inlet venules : from these, circulating blood passes through openings in the limiting plate into hepatic sinusoids and then further flows between the hepatic plates to be collected by the central vein (CV in Fig. 41.1e, f). In a few hepatic lobules, exogenous goat FITC-IgG was only distributed in about half of their areas (Fig. 41.1f), indicating that blood was circulating through the portal lobule. The three-dimensional vascular network revealed by the injected goat FITC-IgG could also be obtained by collecting images of serial sections.
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