Immunohistochemical analysis of various serum proteins in living mouse thymus with "in vivo cryotechnique".

It has been difficult to clarify the precise localizations of soluble serum proteins in thymic tissues of living animals with conventional immersion- or perfusion-fixation followed by alcohol dehydration owing to ischemia and anoxia. In this study, "in vivo cryotechnique" (IVCT) followed by freeze-substitution fixation was performed to examine the thymic structures of living mice and immunolocalizations of intrinsic or extrinsic serum proteins, which were albumin, immunoglobulin G1 (IgG1), IgA, and IgM, as well as intravenously injected bovine serum albumin (BSA). Mouse albumin was more clearly immunolocalized in blood vessels and interstitial matrices of the thymic cortex than in tissues prepared by the conventional methods. The immunoreactivities of albumin and IgG1 were stronger than those of IgA and IgM in the interstitium of subcapsular cortex. The injected BSA was time-dependently immunolocalized in blood vessels and the interstitium of corticomedullary areas at 3.5 h after its injection, and then gradually diffused into the interstitium of the whole cortex at 6 h and 12 h. Thus, IVCT revealed definite immunolocalizations of serum albumin and IgG1 in the interstitium of thymus of living mice, indicating different accessibility of serum proteins from the corticomedullary areas, not from the subcapsular cortex of living animals, depending on various molecular sizes and concentrations.