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

Grote, Monika

doi:10.1002/jemt.1070210307

Cited by 22 publications

(22 citation statements)

References 25 publications

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“…To obtain a signal strong enough to be detected by immunolocalization, other authors have used anhydrous fixation techniques (Grote, 1991(Grote, ,1992Grote et al,, 1994). Although the Ole e I protein of olive pollen is also a low molecular weight, highly water-soluble molecule (Villalba et al, 1993), we were able to detect this allergen after aqueous fixation followed by incubation with an MAb (Rodriguez-Garcia et al, 1995;MartinOrozco et al, 1994).…”

Section: Discussionmentioning

confidence: 73%

Immunogold probes for light and electron microscopic localization of Ole e I in several Oleaceae pollens.

Fernández¹,

Olmedilla²,

Alché³

et al. 1996

J Histochem Cytochem.

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We investigated the immunolocalization of the olive major allergen Ole e I and Ole e I-like proteins in pollen from several Oleaceae species [olive (Olea europaea), ash (Frax~hus exmhior), privet (I;igUsrum dgarik) , lilac (Syringa dgare), and forsythia (Forsythia suspensa)] . Crossreactions among different pollens were found in enzyme immunoassays. For immunolocalization with light microscopy we used the silver enhancement technique with three monoclonal antibodies (1D8, 10H1, and 16G2) that recognize three different epitopes of the allergen Ole e I. Our findings show that the silver enhancement technique is very useful when several

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Section: Discussionmentioning

confidence: 73%

Immunogold probes for light and electron microscopic localization of Ole e I in several Oleaceae pollens.

Fernández¹,

Olmedilla²,

Alché³

et al. 1996

J Histochem Cytochem.

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“…The origin and location of allergenic proteins in pollen has been intensely studied over recent years. Due to the extremely high water solubility of the pollen allergens several research groups have applied techniques of anhydrous fixation (Grote 1992, Grote et al 1993, Staff et al 1990). It has been proved that the allergens of birch occur in the cytoplasm of the pollen grains (Grote et al 1993), and that the allergens of grass pollen appeared in the cytoplasm as well as in the starch grains (Taylor et al 1994).…”

Section: Discussionmentioning

confidence: 99%

Orbicules inBetula Pendulaand their Possible Role in Allergy

El‐Ghazaly¹,

Takahashi²,

Nilsson³

et al. 1995

Grana

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The external morphology, internal structure and stainability for protein of orbicules in Betitla pertdula are examined by SEM and TEM. The pollen and the orbicule walls stain moderately for protein. The protein is localized mainly at the pollen aperture, in a thin layer betneen the exine and the intine and in the core and on the surface of the orbicules. The nitrocellulose membrane test indicates possible allergenicity of the orbicules as well as of the pollen. Since the orbicules of Befirla peiidula are 2-4 pm in diameter, they can pass through the bronchiole of the lungs and cause bronchial asthma. Orbicules have been recognized since 1865 by Rosanoff, who described a membrane with granules around the pollinia in the Mimosaceae. Ubisch (1927) and Kosmath (1927) published lists of different species that contain tapetal cells with orbicules on their surface. These early studies were followed by several others which were based solely on light microscopy investigations (for detailed review, see Banerjee 1967 andRaj &.Later studies using SEM andTEM were published on the morphology and ontogeny of orbicules (Ubisch bodies), e.g., Rowley (1962Rowley ( , 1963 To our knowledge, there is no information published on the morphology, ontogeny and histochemistry of orbicules in Befirla.The allergenicity of Befirla pollen is well documented (Wihl et al. 1988. However the possible allergenic effects of the orbicules in Betrrla have not yet been investigated. Takahashi et a1 (1991, 1995) demonstrated the presence of small particles with Cry j allergen activity together with intact, airborne Cryptoiseria japonica pollen *grains during the pollen season. The sites of C r y j I were found to be on the pollen exine and on the orbicule walls (MikiHirosige et al. 1994 andTakahashi et al. 1992).In the present study we describe the morphology and ultrastructure of orbicules in Betrrla pendirln. The allergenic effects of Betirla pollen and orbicules are also tested by the immunoblotting method. MATERIAL AND METHODSThe material for this study was collected during the period 1989-1993 from a Berula tree growing close to the Palynological Laboratory, Stockholm.For SEM preparations, fresh anthers mere dehydrated in an acetone series. Anthers were opened longitudinally and mounted on a stub. They were coated with gold for 3 minutes and examined with a Jeol JSM 6300 microscope.For TEhl preparations, anthers \\ere futed in 2% glutaraldehyde (GA) in 0.05 M Nacacodylate buffer, pH 7.4 at room temp. for 24 hours, postfixed with 0~01, then dehydrated in an acetone series, embedded in Spurr's medium (Spurr 1969) and sectioned. Ultrathin sections were stained with 1% aqueous uranyl acetate for 5 min, washed with distilled water, and floated on lead citrate stain for 5 minutes in order to improve the general contrast. Stain for protein: Sections on gold grids were stained with 5% phosphotungstic acid (FTA) in 10% acetone for 15min (Benedetti & Bertolini 1963, Marinozzi 1968. Aeroallergen immunoblotting procedureSamples collected on Burkard tapes w...

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“…Olive pollen was dehydrated in a graded series of ethanol and embedded in Lowicryl K4 M at –35°C. Details of the preparation procedures have been published before [23, 24]. After polymerization of the resin-embedded materials, ultrathin sections were cut and placed on nickel grids for immunogold labeling.…”

Section: Methodsmentioning

confidence: 99%

Immunogold Electron Microscopic Localization of the 2 EF-Hand Calcium-Binding Pollen Allergen Phl p 7 and its Homologues in Pollens of Grasses, Weeds and Trees

Grote

Westritschnig

Valenta

2008

Int Arch Allergy Immunol

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Background: The 2 EF-hand calcium-binding allergen from timothy grass pollen, Phl p 7, contains the majority of relevant IgE epitopes among calcium-binding allergens occurring in pollen species of different plants. Objective: To describe the ultrastructural localization of Phl p 7 allergen in timothy grass pollen and its homologues in a broad spectrum of allergologically relevant pollens from grasses (timothy grass, rye grass), trees (birch, alder, olive) and weeds (mugwort, ribwort, ragweed) commonly growing in Europe. Materials and Methods: Mature pollens from 8 different plant species were collected and anhydrously prepared for transmission electron microscopy. In ultrathin sections, allergens were localized using an antibody prepared against a Phl p 7-derived peptide comprising the C-terminal half of the Phl p 7 wild-type molecule in combination with a secondary antibody coupled to 10-nm colloidal gold particles. Results: Phl p 7 and Phl p 7 homologues were detected in pollen from each of the 8 pollen species investigated. The allergens were found in the cytoplasm of the pollen grains (cytoplasmic matrix, mitochondria, nuclei) and in the pollen wall (preferably the exine). Reserve materials were unlabeled. Conclusions: The 2 EF-hand calcium-binding allergen Phl p 7 from timothy grass and its homologues can be localized in all pollen species under investigation. This finding confirms that Phl p 7 is a marker allergen for sensitization of patients to a novel family of 2 EF-hand calcium-binding pollen allergens occurring in a number of important allergenic plants in Europe.

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

Cited by 22 publications

References 25 publications

Immunogold probes for light and electron microscopic localization of Ole e I in several Oleaceae pollens.

Immunogold probes for light and electron microscopic localization of Ole e I in several Oleaceae pollens.

Orbicules inBetula Pendulaand their Possible Role in Allergy

Immunogold Electron Microscopic Localization of the 2 EF-Hand Calcium-Binding Pollen Allergen Phl p 7 and its Homologues in Pollens of Grasses, Weeds and Trees

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