MRT Letter: Application of novel “in vivo cryotechnique” in living animal kidneys

Aim: To compare the influence of different fixation procedures on morphologic studies in living mice, and to identify the advantages of the “in vivo cryotechnique” (IVCT). Methods: We prepared mouse kidneys using four different fixation methods: conventional immersion-fixation, quick-freezing following resection of the kidney, quick-freezing following perfusion-fixation, and IVCT. Results: Kidney glomeruli were noticeably contracted after conventional immersion-fixation or quick-freezing following resection compared to glomeruli from tissues preserved by the IVCT. With the IVCT, both albumin and IgG were colocalized exclusively along or within the glomerular capillary loops; however, immunoreactivity of these proteins in the other three methods was clearly detected in the Bowman's space and apical cytoplasm of the proximal tubules. With the IVCT, immunoreactivity of collagen type IV was very weak at the glomerular basement membrane (GBM) until microwave treatment, which increased its immunoreactivity. In contrast, the immunoreactivity was clearly detected at the GBM with or without microwave treatment with quick-freezing following perfusion-fixation. With quick-freezing following perfusion-fixation, aquaporin-1 (AQP-1) was irregularly distributed in a disorganized manner on the brush border and apical cell membrane along the proximal tubules. But AQP-1 was labeled intensely and regularly along the brush border and apical cell membrane andonly weakly along the basolateral membrane of the proximal tubules with the IVCT. Conclusion: The IVCT may reliably maintain soluble serum proteins and renal intrinsic proteins such as AQP-1 in situ and capture transient structures and functional changes in vivo. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.