Fig. 20.1
(a) A schematic drawing of “in vivo cryotechnique ” for mouse kidneys under acute hypertensive condition . The abdominal aorta is ligated by thin thread just below both renal arteries. A left kidney is directly frozen in vivo with a cryoknife , and the isopentane-propane cryogen (−193 °C) precooled in liquid nitrogen is simultaneously poured over the cryocut kidney under the “in vivo cryoapparatu s ,” as shown in (b). (c) After the “in vivo cryotechnique ,” the frozen specimen is processed for immunohistochemistry , as following in the flowchart
Then the “in vivo cryotechnique ” was performed under such various blood flow conditions as described in the previous paragraph. Briefly, a cryoknife edge precooled in liquid nitrogen (−196 °C) was positioned over the left kidney (Fig. 20.1a). The mouse kidney was cut with the cryoknife edge, and liquid isopentane -propane cryogen (−193 °C) was simultaneously poured over it with an assistance of the “in vivo cryoapparatu s ” (VI-11; Eiko Engineer Co. Ibaraki, Japan) (Fig. 20.1b), which had been already invented for the purpose of “in vivo cryotechnique ” [7]. The frozen kidneys were carefully trimmed out with a dental drill in liquid nitrogen and then processed for the following freeze-substitution step (Fig. 20.1c), as reported before [1–3].
20.3 Immunolocalization of Albumin and IgG in Renal Proximal Tubules
To examine the glomerular leakage and reabsorption of serum protein s in renal proximal tubule s , the immunohistochemistry for albumin and IgG was performed on paraffin sections prepared by the “in vivo cryotechnique ” (Fig. 20.2). Under the acute hypertensive condition , however, in addition to the similar immunolocalization under the normotension, both albumin (Fig. 20.2b, c) and IgG (Fig. 20.2e, f) were clearly immunolocalized along cell membranes containing apical brush borders and also in the apical cytoplasm of most proximal tubule s (Fig. 20.2c, f).
Fig. 20.2
Immunohistochemical localization of albumin (a–c) and IgG (d–f) in mouse kidneys under the acute hypertensive condition , as revealed by the “in vivo cryotechnique ” (a and d, HE staining; b and e, peroxidase-DAB; c and f, higher magnified image). Under the acute hypertension , the immunolocalization of both albumin and IgG is clearly observed along the basolateral cell membranes and also in the apical cytoplasm of almost all proximal tubule s (b, c, e, f). Scale bar 20 μm
20.4 Immunolocalization of IgG Light or Heavy Chains in Proximal Tubules
To examine whether IgGs with the full molecular length were leaked out through glomerular capillary loop s under the acute hypertensive condition , another immunostaining analysis for the IgG light or heavy chain was performed, respectively, on serial paraffin sections (Fig. 20.3). In the cortex of mouse kidneys under such acute hypertension , both kappa (Fig. 20.3b) and lambda (date not shown) light chains’ immunolabeling was clearly detected along the apical cell membranes of almost all proximal tubule s , in the similar way to those revealed with the antibody against the full-length IgG described in the previous section. To the contrary, the antibody against the IgG1 heavy chain was not immunoreacted in such proximal tubular areas, but in the blood vessel s and the interstitium (Fig. 20.3c).
Fig. 20.3
Light micrographs of mouse renal cortices on serial sections by HE staining (a) and immunolabeled by two kinds of antibodies against kappa light chain (b) and IgG1 heavy chain (c) under the acute hypertensive condition . Strong immunolabeling of the kappa light chain is observed in the apical cytoplasm of almost all proximal tubule s (b). To the contrary, the IgG1 heavy chain is not seen to be localized in the apical cytoplasm of the proximal tubules (c), but in the glomerular blood capillaries and blood vessel s in the interstitium between the renal tubules. Scale bar 20 μm
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
