Abstract
To show the three-dimensional distribution of proteins in renal cells, we applied the immunogold scanning electron microscopy method using vibratome slices. Kidney specimens from puromycin aminonucleoside (PAN) nephrotic rats and controls were obtained after intravenous infusion of human serum albumin and fixed in periodate-lysine-paraformaldehyde solution. Vibratome slices were incubated with anti-human albumin antibody and 25-nm gold-labeled secondary antibody. After silver enhancement, the immunogold particles were clearly observed by backscatter electron imaging, whereas they were ambiguous in the secondary electron image. The images showed a higher resolution of the tissues at an acceleration voltage of 5 mV than at 10 mV. Immunogoldlabeled albumin was observed in the lumen and endocytotic vesicles of the proximal tubules, and on the podocyte surface in the PAN nephrotic rats, whereas only a few particles were observed in the controls. In conclusion, silver-enhanced immunogold scanning electron microscopy at low acceleration voltages using vibratome sections can be applicable for detecting the intracellular/extracellular localization of molecules in solid tissues. We succeeded in visualizing the enhanced albumin endocytosis of the proximal tubules and the exocytosis of albumin from podocytes in the nephrotic rats.
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Faulk WP, Taylor GM (1971) An immunocolloid method for the electron microscope. Immunochemistry 8:1081–1083
Beil M, Micoulet A, von Wichert G, Paschke S, Walther P, Omary MB, Van Veldhoven PP, Gern U, Wolff-Hieber E, Eggermann J, Waltenberger J, Adler G, Spatz J, Seufferlein T (2003) Sphingosylphosphorylcholine regulates keratin network architecture and visco-elastic properties of human cancer cells. Nat Cell Biol 5:80–811
Biggs MJ, Richards RG, Wilkinson CD, Dalby MJ (2008) Focal adhesion interactions with topographical structures: a novel method for immuno-SEM labelling of focal adhesions in S-phase cells. J Microsc 231:28–37
Cai Y, Gao Y, Sheng Q, Miao S, Cui X, Wang L, Zong S, Koide SS (2002) Characterization and potential function of a novel testisspecific nucleoporin BS-63. Mol Reprod Dev 61:126–134
de Harven E, Leung R, Christensen H (1984) A novel approach for scanning electron microscopy of colloidal gold-labeled cell surfaces. J Cell Biol 99:53–57
Erlandsen SL, Bemrick WJ, Schupp DE, Shields JM, Jarroll EL, Sauch JF, Pawley JB (1990) High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging. J Histochem Cytochem 38:625–632
Gross DK, De Boni U (1990) Colloidal gold labeling of intracellular ligands in dorsal root sensory neurons, visualized by scanning electron microscopy. J Histochem Cytochem 38:775–784
Iwano M, Che FS, Takayama S, Fukui K, Isogai A (2003) Threedimensional architecture of ribosomal DNA within barley nucleoli revealed with electron microscopy. Scanning 25:257–263
Kiseleva E, Drummond SP, Goldberg MW, Rutherford SA, Allen TD, Wilson KL (2004) Actin- and protein-4.1-containing filaments link nuclear pore complexes to subnuclear organelles in Xenopus oocyte nuclei. J Cell Sci 117:2481–2490
Kitamura K, Suganuma N, Takata K, Matsuyama K, Goto J, Furuhashi M, Kanayama N (2003) Changes in oligosaccharide expression on plasma membrane of the mouse oocyte during fertilisation and early cleavage. Zygote 11:183–189
Lea P, Lee LM, Shi QW, Takahashi M, Youn W, Jackowski G (1996) Advantages of backscatter electron imaging scanning electron microscopy for intracellular localization of cardiac analytes by gold conjugated antibody. Scanning 18:259–268
Lee A, White N, van der Walle CF (2003) The intestinal zonula occludens toxin (ZOT) receptor recognises non-native ZOT conformers and localises to the intercellular contacts. FEBS Lett 555:638–642
Madore N, Smith KL, Graham CH, Jen A, Brady K, Hall S, Morris R (1999) Functionally different GPI proteins are organized in different domains on the neuronal surface. EMBO J 18:6917–6926
Schwarzer C, Becker S, Awni LA, Cole T, Merker R, Barnikol-Watanabe S, Thinnes FP, Hilschmann N (2000) Human voltagedependent anion-selective channel expressed in the plasmalemma of Xenopus laevis oocytes. Int J Biochem Cell Biol 32:1075–1084
Goldberg MW (2008) Immunolabeling for scanning electron microscopy (SEM) and field emission SEM. Methods Cell Biol 88:109–130
Ferguson DJ, Burns J, Harrison D, Jonasson JA, McGee JO (1986) Chromosomal localization of genes by scanning electron microscopy using in situ hybridization with biotinylated probes: Y chromosome repetitive sequences. Histochem J 18:266–270
de Souza W, Campanati L, Attias M (2008) Strategies and results of field emission scanning electron microscopy (FE-SEM) in the study of parasitic protozoa. Micron 39:77–87
Goldberg MW, Allen TD (1992) High-resolution scanning electronmicroscopy of the nuclear-envelope: demonstration of a new, regular, fibrous lattice attached to the baskets of the nucleoplasmic face of the nuclear-pores. J Cell Biol 119:1429–1440
Kluck RM, Esposti MD, Perkins G, Renken C, Kuwana T, Bossy-Wetzel E, Goldberg M, Allen T, Barber MJ, Green DR, Newmeyer DD (1999) The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol. J Cell Biol 147:809–822
Long HA, Boczonadi V, McInroy L, Goldberg M, Maatta A (2006) Periplakin-dependent re-organisation of keratin cytoskeleton and loss of collective migration in keratin-8-downregulated epithelial sheets. J Cell Sci 119:5147–5159
Suzuki E (2002) High-resolution scanning electron microscopy of immunogold-labelled cells by the use of thin plasma coating of osmium. J Microsc 208:153–157
Albrecht RM, Prudent J, Simmons SR, Pawley J, Choate JJ (1989) Observations of colloidal gold labelled platelet microtubules: high voltage electron microscopy and low voltage-high resolution scanning electron microscopy. Scanning Microsc 3:273–278
Pawley JB, Erlandsen SL (1989) The case for low voltage high resolution scanning electron microscopy of biological samples. Scanning Microsc Suppl 3:163–178
Autrata R (1989) Backscattered electron imaging using singlecrystal scintillator detectors. Scanning Microsc 3:739–763
Schauer P, Autrata R (1992) Light transport in single-crystal scintillation detectors in SEM. Scanning 14:325–333
Hermann R, Muller M (1992) Towards high-resolution SEM of biological objects. Arch Histol Cytol 55:17–25
Deharven E, Soligo D (1986) Scanning electron-microscopy of cell-surface antigens labeled with colloidal gold. Am J Anat 175:277–287
Erlandsen SL, Frethem C, Autrata R (1990) Workshop on highresolution immunocytochemistry of cell surfaces using field emission SEM. J Histochem Cytochem 38:1779–1780
Scopsi L, Larsson LI (1986) Colloidal gold probes in immunocytochemistry. An optimization of their application in light microscopy by use of silver intensification procedures. Med Biol 64:139–145
Yu Y, Leng CG, Terada N, Ohno S (1998) Scanning electron microscopic study of the renal glomerulus by an in vivo cryotechnique combined with freeze-substitution. J Anat 192:595–603
Madsen KM, Zhang L, Abu Shamat AR, Siegfried S, Cha JH (1997) Ultrastructural localization of osteopontin in the kidney: induction by lipopolysaccharide. J Am Soc Nephrol 8:1043–1053
Schlingemann RO, Rietveld FJ, de Waal RM, Ferrone S, Ruiter DJ (1990) Expression of the high molecular weight melanomaassociated antigen by pericytes during angiogenesis in tumors and in healing wounds. Am J Pathol 136:1393–1405
Tojo A, Gross SS, Zhang L, Tisher CC, Schmidt HH, Wilcox CS, Madsen KM (1994) Immunocytochemical localization of distinct isoforms of nitric oxide synthase in the juxtaglomerular apparatus of normal rat kidney. J Am Soc Nephrol 4:1438–1447
Yang CW, Li C, Jung JY, Shin SJ, Choi BS, Lim SW, Sun BK, Kim YS, Kim J, Chang YS, Bang BK (2003) Preconditioning with erythropoietin protects against subsequent ischemia-reperfusion injury in rat kidney. FASEB J 17:1754–1755
Tanaka A (1994) Osmium conductive metal-coating for SEM specimen using sublimated osmium-tetroxide in negative glow phase of DC glow-discharge. J Electron Microsc 43:177–182
Peters KR (1986) Rationale for the application of thin, continuous metal-films in high magnification electron-microscopy. J Microsc 142:25–34
Tojo A, Endou H (1992) Intrarenal handling of proteins in rats using fractional micropuncture technique. Am J Physiol 263:F601–F606
Tencer J, Frick IM, Oquist BW, Alm P, Rippe B (1998) Sizeselectivity of the glomerular barrier to high molecular weight proteins: upper size limitations of shunt pathways. Kidney Int 53: 709–715
Tojo A, Onozato ML, Kitiyakara C, Kinugasa S, Fukuda S, Sakai T, Fujita T (2008) Glomerular albumin filtration through podocyte cell body in puromycin aminonucleoside nephrotic rat. Med Mol Morphol 41:92–98
Tojo A, Onozato ML, Ha H, Kurihara H, Sakai T, Goto A, Fujita T, Endou H (2001) Reduced albumin reabsorption in the proximal tubule of early-stage diabetic rats. Histochem Cell Biol 116:269–276
Tojo A, Onozato ML, Kurihara H, Sakai T, Goto A, Fujita T (2003) Angiotensin II blockade restores albumin reabsorption in the proximal tubules of diabetic rats. Hypertens Res 26:413–419
Russo LM, Sandoval RM, McKee M, Osicka TM, Collins AB, Brown D, Molitoris BA, Comper WD (2007) The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: retrieval is disrupted in nephrotic states. Kidney Int 71: 504–513
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Kinugasa, S., Tojo, A., Sakai, T. et al. Silver-enhanced immunogold scanning electron microscopy using vibratome sections of rat kidneys: detection of albumin filtration and reabsorption. Med Mol Morphol 43, 218–225 (2010). https://doi.org/10.1007/s00795-010-0500-9
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DOI: https://doi.org/10.1007/s00795-010-0500-9