Conventional immunohistochemical techniques have some problems of molecular diffusion artifact and antigen masking for the detection of immunoglobulin in animal organs, which were already reported in large malignant cells like the Reed-Sternberg cells of Hodgkin’s lymphoma [18]. In the present study, the diffusion artifacts of both albumin and IgG1 were also observed in tumor cells prepared by FQF (Fig. 50.2). However, such technical artifact s can be mostly prevented by IVCT and Cb, because cells and tissues in target organs of living animal bodies are immediately cryofixed at the time of freezing in situ.

The time-dependent immunolocalization of injected BSA was revealed within the tumor tissues, as shown in Fig. 50.3. The injected BSA was mostly immunolocalized within blood vessel s in the tumor mass and the capsular connective tissue 10 min after the injection, but later it was diffusely immunolocalized in the surrounding connective tissue and a fraction of the interstitium in the tumor mass. Some previous studies reported that BSA leaked out into the interstitial spaces within 1 h, using live imaging with intravital or a confocal laser scanning microscope [8, 19]. Small amounts of molecules may be difficult to visualize on histological sections by immunohistochemistry . However, because different intensities of immunoreactivity in a single histological section usually depend on the relative amount of active antigen sites, our findings suggest that the permeability of blood vessels in the tumor tissue is distinct from that in the surrounding connective tissue of the tumor capsule, which would partly depend on the molecular structure of blood vessels [16, 20]. In fact, we have detected abundant immunolocalization of BSA in the connective tissue of the tumor capsules 24 h after the BSA injection. In addition, 72 h after the BSA injection, BSA immunolocalization was more clearly detected in the interstitium of the tumor tissue. The repeated injection of BSA induced the accumulation of BSA in the blood circulation , thus causing the prolonged exposure of the tumor mass to higher concentrations of BSA over a few days, resulting in the increased concentration of leaked BSA in the tumor interstitium.