Distribution of serum protein s in mouse liver was also clearly detected with IVCT [11]. Under normal blood circulation , immunostaining for IgG1 , Igκ, or albumin was observed along sinusoidal edges (Fig. 5.2). Igκ immunostaining also represents IgA and was found in bile canaliculi located in hepatic plates visualized by tight junction s immunopositive for ZO-1. Albumin immunoreactivity appeared as small dots and was observed in hepatocytes , presumably detected in Golgi areas. In tissue specimens prepared with PF-DH, immunostaining for serum proteins including Igκ and albumin was less clearly observed (Fig. 5.2).

Soluble glycogen in hepatocytes was also well maintained with IVCT [11]. Abundant glycogen as visualized by strong periodic acid-Schiff (PAS ) staining was detected in hepatocytes located around centrilobular sinusoids and periportal zones with IVCT under normal blood circulation . In contrast, by FQF, the PAS staining was partially decreased in the peripheral cytoplasm of hepatocytes around centrilobular sinusoids. By PF-DH, such loss of PAS staining was more clearly observed in periportal zones, and PAS staining intensity in the cytoplasm was decreased in some hepatocytes located in the centrilobular areas. Decrease of PAS staining was worst in the liver specimens prepared by IM-DH.

Soluble molecules can be moved or lost in conventional tissue preparation methods such as PF-DH or IM-DH [29–32]. However, IVCT and QF do not use aqueous fixatives during specimen preparation and thus well-preserve soluble component s in the specimens [29, 31–33]. Signal molecules influencing hepatic microvasculature have been studied under pathological conditions [5–7, 34]. The nutritional state, such as the glycogen storage of hepatocytes , could be important for injury caused by ischemia -reperfusion stress during liver transplantation [35]. Therefore, the localization of soluble molecules would be effectively examined in living animal livers using IVCT.

Because of the excellent preservation of soluble molecules in situ by the quick freezing followed by freeze-substitution, these methods also revealed that immunoreactivity of serum protein s is detected in some hepatocyte cytoplasm after tissue resection (Fig. 5.3) [11]. Interestingly, immunostaining for IgG1 and Igκ in the serial sections demonstrated that these serum proteins were colocalized in the same hepatocyte. The IgG1- and Igκ-immunopositive hepatocytes were also immunopositive for albumin , while some hepatocytes were immunopositive only for albumin (Fig. 5.3). Such diffuse immunostaining for the serum proteins was not detected with IVCT [11]. Since the molecular size of IgG is larger than that of albumin, the immunoreactivity of serum proteins in hepatocytes indicated size-dependent translocation of soluble serum protein s due to rapid permeability alterations of cell membranes caused by tissue resection. Actually, the disruption of the cell membranes is a hallmark of necrosis [36]. Soluble serum proteins as well as extrinsic dye would be easily translocated into hepatocytes upon necrosis which is characterized by size-dependent changes of cell membrane permeability following anoxia /ischemia . IVCT could be useful to analyze the integrity of hepatocyte cell membranes under physiological and pathological conditions in living animal liver .