Application of periodic acid-Schiff fluorescence emission for immunohistochemistry of living mouse renal glomeruli by an "in vivo cryotechnique".

Abstract

To identify the distribution of endogenous serum proteins in living mouse renal glomeruli under various hemodynamic conditions, we used the periodic acid-Schiff (PAS) and its fluorescence emission as a marker for the glomerular basement membrane (GBM). The immunostaining for collagen type IV was hardly observed without microwave treatment in specimens prepared by an "in vivo cryotechnique". However, PAS staining and its fluorescence emission could be clearly visualized at the GBM with the "in vivo cryotechnique". Under normotensive conditions, immunoreaction products of albumin and immunoglobulin G heavy and light chains (IgG(H+L)) were localized within glomerular capillary loops (GCL) but not colocalized with the PAS fluorescence emission of the GBM. Under heart-arrest conditions and with quick-freezing of resected tissues, albumin, IgG (H+L), immunoglobulin kappa light chain, and IgG1 heavy chain (IgG1) were immunolocalized within the GCL and mesangial areas, but only albumin and the kappa light chain were additionally immunolocalized in Bowman's space, indicating their passage through the GBM. Under acute hypertensive conditions, both albumin and the kappa light chain, but not IgG1, were clearly immunolocalized along the GBM and in the Bowman's space, indicating their increased passage through the GBM. The overlapping areas of PAS fluorescence emission and the albumin or kappa light chain appeared to be larger with quick-freezing and under the heart arrest or acute hypertensive conditions than under normal circulation, whereas those of PAS emission and IgG1 did not differ among these conditions. The serum proteins passing through the GBM were clearly visualized with the "in vivo cryotechnique", immunofluorescence staining, and PAS fluorescence emission.