Summary
The influence of fixation and the embedding medium on post-embedment dialyzed iron (DI) staining of acidic complex carbohydrates in mouse colon was studied at the ultrastructural level. DI staining of ultrathin sections from non-osmicated tissues embedded in epoxy resins was very weak, whereas DI staining of non-osmicate tissues embedded in non-epoxy resins such as polystyrene, polyester resins, methacrylates and newly developed embedding mixtures was strong. Brief exposure to OsO4 (5 min) abolished the DI staining in stored secretions of goblet and mucous cells, in apical cytoplasmic vesicles and at the microvillous surface of columnar absorptive cells and in the Golgi cisternae of all colonic cell types in epoxy embedded tissues, but only reduced slightly or had no effect on the DI reactivity observed in these sites in tissues embedded in non-epoxy resins. Prolonged exposure to OsO4 (60 min) prior to embedment in non-epoxy resins further reduced DI staining in all reactive sites and abolished the staining in Golgi cisternae of all colonic epithelial cells. Embedment of non-osmicated tissues in a styrene-Vestopal W mixture and of tissues briefly exposed to OsO4 after primary glutaraldehyde fixation in styrene-methacrylate is recommended for optimal post-embedment DI staining of the acidic groups of complex carbohydrates.
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References
Berlin JD (1967) The localization of acid mucopolysaccharides in the Golgi complex of intestinal goblet cells. J Cell Biol 32:760–766
Blanquet PR, Loiez A (1974) Colloidal iron used at pH's lower than 1 as electron stain for surface proteins. J Histochem Cytochem 22:368–377
Courtoy R, Boniver J, Simar LJ (1974) A cetylpyridinium chloride (CPC) and ferric thiocyanate (FeTh) method for polyanions demonstration on thin sections for electron microscopy. Histochemistry 42:133–139
Curran RC, Clark AE (1964) The use of the colloidal-iron method for acid mucopolysaccharides in electron microscopy. Biochem J 90:2P
Curran RC, Clark AE, Lovell D (1965) Acid mucopolysaccharides in electron microscopy — the use of the colloidal iron method. J Anat 99:427–434
Estes LW, Apicella JV (1969) A rapid embedding technique for electron microscopy. Lab Invest 20:159–173
Francioni G, Borgioli G (1979) Polystyrene embedding: A new method for light and electron microscopy. Stain Technol 54:167–172
Gasic GJ, Berwick L, Sorrentino M (1968) Positive and negative colloidal iron as cell surface electron stains. Lab Invest 18:63–71
Geyer G, Stibenz D (1974) Quantitative aspects of the colloidal iron reaction. Acta Histochem 50:264–272
Hardin JH, Spicer SS (1971) Ultrastructural localization of dialyzed iron-reactive mucosubstance in rat heterophils, and eosinophils. J Cell Biol 48:368–386
Kushida H (1960) A new polyester embedding method for ultrathin sectioning. J Electron Microsc (Oxford) 9:113–116
Kushida H (1961) A styrene-methacrylate resin embedding method for ultrathin sectioning. J Electron Microsc (Oxford) 10:16–19
Lai M, Lampert IA, Lewis PD (1975) The influence of fixation on staining of glycosaminoglycans in glial cells. Histochemistry 41:275–279
Luft JH (1961) Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol 9:409–414
Matukas VJ, Panner BJ, Obison JL (1967) Studies on ultrastructural identification and distribution of protein-polysaccharide in cartilage matrix. J Cell Biol 32:365–377
Mohr WP, Cocking EC (1968) A method for preparing highly vacuolated, senescent or damaged plant tissue for ultrastructural study. J Ultrastruct Res 21:171–181
Pousty I, Bari-Khan MA, Butler WF (1975) Leaching of glycosaminoglycans by the fixatives formalin-saline and formalin-cetrimide. Histochem J 7:361–365
Pratt RM, Larsen MA, Johnston MC (1975) Migration of cranial neural crest cells in a cell free, hyaluronate-rich matrix. Dev Biol 44:298–305
Rambourg A (1974) Staining of Intracellular Glycoproteins. In: Wisse E, Daems WT, Molenaar I, van Duijn P (eds) Electron microscopy and cytochemistry. North-Holland, Amsterdam London, pp 245–253
Revel JP (1964) A stain for ultrastructural localization of acid mucopolysaccharides. J Microsc (Paris) 3:535–544
Ryter A, Kellenberger E (1958) L'inclusion au polyester pour l' ultramicrotomie. J Ultrastruct Res 2:200–214
Sannes PL, Spicer SS, Katsuyama T (1979) Ultrastructural localization of sulfated complex carbohydrates with a modified iron diamine procedure. J Histochem Cytochem 27:1108–1111
Sato A, Spicer SS (1980) Ultrastructural cytochemistry of complex carbohydrates of gastric epithelium in the guinea pig. Am J Anat 159:307–329
Shinagawa Y, Ogura M (1960) Polystyrene embedding method for ultrathin sectioning. J Electron Microsc (Oxford) 9:148–150
Spicer SS, Staley MW, Wetzel MG, Wetzel BK (1967) Acid mucosubstances and basic protein in mouse Paneth cells. J Histochem Cytochem 15:225–242
Spicer SS, Katsuyama T, Sannes PL (1978) Ultrastructural carbohydrate cytochemistry of gastric epithelium. Histochem J 309–331
Spicer SS, Mochizuki, Setser ME, Martinez JR (1980) Complex carbohydrates of rat tracheobronchial surface epithelium visualized ultrastructurally. Am J Anat 158:93–109
Spurr AR (1969) A low viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43
Sturgess IM, Mitranic MM, Moscarello MA (1978) Extraction of glycoproteins during tissue preparation for electron microscopy. J Microsc (Oxford) 114:101–105
Tadano Y, Yamada K (1979) Ultrastructural features of acidic complex carbohydrates in the intercellular matrix of the ovarian follicles in adult mice. Histochemistry 60:125–133
Takamiya H, Batsford S, Vogt A (1980) An approach to postembedding staining of protein (immunoglobulin) antigen embedded in plastic: Prerequisites and limitations. J Histochem Cytochem 28:1041–1049
Thiéry JP (1970) Cytochimie sur coupe fine des mucopolysaccharides acide apres inclusion dans le resines epoxy. Microscopie Electronique 1970, Proc Intern Congr Electron Micr, Grenoble, 1:577–578
Thiéry JP, Ovtracht L (1979) Differential characterization of carboxyl and sulfate groups in thin sections for electron microscopy. Biol Cell 37:281–288
Thomopoulos GN, Schulte BA, Spicer SS (1983) The influence of embedding media and fixation on the post-embedment ultrastructural demonstration of complex carbohydrates. I. Morphology and periodic acid-thiocarbohydrazide-silver proteinate staining of vicinal diols. Histochem J. 15:763–784
Weinstock M, Bonneville MA (1971) Compartments rich in acidic carbohydrate-protein complexes within electrolyte- and water-transporting cells. Lab Invest 24:355–367
Wetzel MG, Wetzel BK, Spicer SS (1966) Ultrastructural localization of acid mucosubstances in the mouse colon with iron-containing stains. J Cell Biol 30:299–315
Yamada K (1973) Cytochemistry (Polysaccharides). In: Textbook of techniques for electron microscopy (Nagoya). Koketsu-shuppan Printing Co, Nagoya, pp 134–147
Yamada K (1974) Acid mucosaccharide-containing structures in the gall bladder epithelium of the rabbit as seen with the electron microscope. Histochemistry 37:351–360
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This research was supported by NIH Grant AM-10956, National Research Service Award GM-08745 and United Medical and Health Foundation of South Carolina, Inc. Grant No. 79
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Thomopoulos, G.N., Schulte, B.A. & Spicer, S.S. The influence of embedding media and fixation on the post-embedment ultrastructural demonstration of complex carbohydrates. Histochemistry 79, 417–431 (1983). https://doi.org/10.1007/BF00491777
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DOI: https://doi.org/10.1007/BF00491777