Immunogold electron microscopy of soluble proteins: localization of Bet v I major allergen in ultra-thin sections of birch pollen after anhydrous fixation techniques

To localize the highly water-soluble major allergen Bet v I in ultra-thin sections of birch pollen, pollen grains were cracked, air-dried, and processed for electron microscopy using one of the following preparation techniques: fixation in aqueous p-formaldehyde + cetylpyridinium chloride; fixation in p-formaldehyde vapor; fixation in benzoquinone vapor; inert dehydration; or no fixation. Afterwards the pollen grains were embedded in Lowicryl K4M resin at low temperature. Ultra-thin sections were cut and incubated with a monoclonal antibody against Bet v I, followed by a gold-labeled secondary antibody. In some experiments, commercial rabbit IgG antibodies against birth pollen allergens were also used, followed by incubation with the protein A-gold complex. Bet v I could be localized only after vapor fixation and in the inert dehydrated specimens. Best preservation of ultrastructure and antigenicity was obtained after p-formaldehyde vapor fixation. Bet v I antibody binding sites were detected only in the cytoplasmic matrix of the pollen grain, never in the pollen wall. Commercial rabbit antibodies bound to cytoplasm and wall of all prepared specimens, even after aqueous fixation. This might be explained by the assumption that these antibodies recognize a variety of antigenic and allergenic structures, not all of which are so highly soluble as Bet v I.     

Electron and X-ray microscopic analyses of reaggregated materials obtained after fractionation of dissolved sporopollenin

Summary Exines fromTypha angustifolia L. pollen were dissolved in hot 2-aminoethanol. The solubilisate was successively fractionated and reaggregated via a dialysis cascade with dialysis tubings of different exclusion volumina. Four fractions of reaggregated material with different molecular mass were obtained. Fraction 1 with a molecular mass above 25,000 Da, fraction 2 with a molecular mass between 10,000–25,000 Da, fraction 3 with a molecular mass between 5,000–10,000 Da, and fraction 4 of a molecular mass lower than 5,000 Da. The fractions were comparatively analysed by scanning and transmission electron microscopy and X-ray microscopy. The material of the fractions with a molecular mass above 10,000 Da exhibit high congruence to the initial material. Analysis of the reaggregated material with the lowest molecular mass revealed special distinct substructures which in form and size showed high similarities to substructures of exines described in literature. In detail, spherical substructures consisting of an electron-dense core surrounded by an electron-transparent corona and in addition elongated substructures with a distinctive surface sculpture were detected.Abbreviations SEM scanning electron microscopy - TEM transmission electron microscopy     

Zur Ornis Nordkameruns

Ohne Zusammenfassung     

Ueber einige Spechte von Adamaua

Ohne Zusammenfassung