The use of cetylpyridinium chloride to preserve water-soluble surface material in pollen for electron microscopy

Grote, Monika; Fromme, Hans Georg; Sinclair, N. J.

doi:10.1016/0047-7206(83)90028-6

Cited by 7 publications

(6 citation statements)

References 9 publications

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“…Sections were stained with uranyl acetate and lead citrate and observed using a Siemens 102 transmission electron microscope. By comparison of the classically fixed material with the CPC fixation, the location of acidic carbohydrates and glycoproteins can be determined as they precipitate and are highly osmiophilic after fixation with CPC (Clarke et al 1980;Grote et al 1983).…”

Section: Methodsmentioning

confidence: 99%

“…Vesicles can be seen between the plasmalemma and the cell wall, and material of similar electron density can be seen outside the cell wall. As these electron-dense bodies cannot be visualised with other fixation procedures, it is concluded that they contain acidic carbohydrates or glycoproteins (Clarke et al 1980;Grote et al 1983).…”

Section: Secretory Role Of the Detached Stigmatic Cellsmentioning

confidence: 99%

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Fine structure of the wet, detached cell stigma of the orchid Dendrobium speciosum Sm.

Slater

Calder

1990

Sexual Plant Reprod

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The stigmatic surface of the orchid Dendrobium speciosum is a cup containing detached cells suspended in a mainly carbohydrate mucilage. The fine structure of the detached cells and their organelles is indicative of secretory cells. The cells contain numerous mitochondria with well-developed cristae, dictyosomes containing extensive cisternae, an extensive network of rough and smooth endoplasmic reticulum and free polysomes throughout. There are many amyloplasts in the vicinity of the nucleus. Vesicles are seen arising from the dictyosomes and endoplasmic reticulum. The plasmalemma is undulating, and vesicles can be seen in its vicinity, giving the typical appearance of a granulocrine secretory system. Cetylpyridinium chloride (CPC) fixation to immobilise acidic carbohydrates detected a highly electron-opaque layer surrounding each cell and globules dispersed through the cell wall. The walls of the detached cells show irregular surface projections which are the remains of pitfields. Biochemical analysis showed that carbohydrates and arabinogalactan proteins are major components of the mucilage.

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Section: Methodsmentioning

confidence: 99%

Section: Secretory Role Of the Detached Stigmatic Cellsmentioning

confidence: 99%

Fine structure of the wet, detached cell stigma of the orchid Dendrobium speciosum Sm.

Slater

Calder

1990

Sexual Plant Reprod

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“…Grote et al (1983Grote et al ( , 1988 and Grote & Fromme (1984a, b) attempted to localize the allergens in birch pollen by precipitation with cetylpyridinium chloride in glutaraldehyde. However, the fixation procedure was aqueous and movement of water soluble allergens was likely before precipitation, although these workers claim to have prevented diffusion of proteins highly soluble in water, and glycoproteins from pollen grains.…”

Section: Introductionmentioning

confidence: 99%

Cellular localization of water soluble, allergenic proteins in rye-grass (Lolium perenne) pollen using monoclonal and specific IgE antibodies with immunogold probes

et al. 1990

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A postembedding method has been developed for localizing water soluble allergens in rye-grass pollen. This uses dry fixation in glutaraldehyde vapour, followed by 2,2-dimethoxypropane, prior to a 100% ethanol series leading into embedment in LR Gold. This has allowed the attachment of specific monoclonal antibodies to the allergen, which are themselves probed with specific immunogold labels to the antibodies. Wall and cytoplasmic sites have been identified, representing an improvement of fixation and localization of allergens over previous studies employing polyclonal, broad spectrum antibodies. Rye-grass allergens are labelled in mature pollen grains in the exine (tectum, nexine and central chamber), and in the electron opaque areas of the cytoplasm, especially mitochondria. The allergens are absent from the intine, polysaccharide (P) particles, amyloplasts, Golgi bodies and endoplasmic reticulum. IgE antibodies derived from humans allergic to rye-grass pollen, bind to similar sites in the cytoplasm but only to the outer surface of the pollen grain wall. This method now provides a valuable tool for further developmental studies on the pollen grains, in order to establish the site/s of synthesis of the allergens.

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“…Soluble proteins are displaced and eventually lost (Hallam, 1976). Therefore, alternative fixation strategies have been invented: 1) non-aqueous fixation in anhydrous fluids such as dimethylsulfoxide or glycerol (Hallam, 1976); 2) vapor fixation using fixatives with a sufficiently high vapor pressure such as osmium tetroxide (Hallam, 1976;Heslop-Harrison, 1979), p-formaldehyde (Eranko, 1964(Eranko, , 1967Frederik et al, 1984;Grote, 1991;Hayat and Giaquinta, 1970; Pearse and Polak, 1975;Pekki and Tuohimaa, 1989), benzoquinone (Pearse and Polak, 1974;Pekki and Tuohimaa, 1989), and glutaraldehyde (Staff et al, 1990); 3) aqueous fixation in the presence of precipitating compounds such as the detergent cetylpyridinium chloride (Grote et al, 1983; Grote, 1989) or the phthalocyanine dye cuprolinic blue (Grote, 1989, Volker et al, 1986; and 4) cryotechniques such as freeze-drying (Vithanage et al, 1980(Vithanage et al, , 1982 or freeze-substitution (Howlett et al, 1973;Martinez and Wick, 1991).…”

Section: Introductionmentioning

confidence: 99%

Ultrastructural morphology and allergen detection in birch pollen after aqueous, anhydrous‐liquid, and vapor fixation techniques

Grote

1992

Microscopy Res & Technique

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In order to find a good compromise between preservation of ultrastructural morphology and retention of antigenicity, birch pollen grains were chemically fixed in aqueous p-formaldehyde or glutaraldehyde, in p-formaldehyde or glutaraldehyde dissolved in anhydrous glycerol, and in p-formaldehyde or glutaraldehyde vapor. Representative cytoplasmic areas were inspected for the preservation of ultrastructural morphology and for their capacity to bind a monoclonal antibody against Bet v I, the major birch pollen allergen. The experiments showed that cytoplasmic morphology was best preserved after vapor fixation in p-formaldehyde. This fixation also led to the highest degree of specific antibody binding.

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The use of cetylpyridinium chloride to preserve water-soluble surface material in pollen for electron microscopy

Cited by 7 publications

References 9 publications

Fine structure of the wet, detached cell stigma of the orchid Dendrobium speciosum Sm.

Fine structure of the wet, detached cell stigma of the orchid Dendrobium speciosum Sm.

Cellular localization of water soluble, allergenic proteins in rye-grass (Lolium perenne) pollen using monoclonal and specific IgE antibodies with immunogold probes

Ultrastructural morphology and allergen detection in birch pollen after aqueous, anhydrous‐liquid, and vapor fixation techniques

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