Preparation of Insect Chromosomes for Immunolabeling

Wolf, Klaus; Glatzel, Sabine; Niedereichholz, F.; Turner, Bryan M.

doi:10.3109/10520299609117150

Cited by 2 publications

(1 citation statement)

References 37 publications

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“…We tested both variants in our experiments and the "wet" approach was clearly better. Similar results have been published recently for preparing insect chromosomes for immunolabeling (Wolf et al 1996). Once the plant protoplasts had dried on the slide, they never yielded a reproducible anti-histone signal because cytoplasmic proteins irreversibly covered the chromosomes and prevented good antibody penetration.…”

Section: Resultssupporting

confidence: 77%

A Cytospin Technique for Spreading Plant Metaphases Suitable for Immunofluorescence Studies

Hladilová

Široký

Vyskot

1998

Biotechnic & Histochemistry

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Recent immunofluorescence techniques enable the localization of various cellular antigens, thus providing a powerful tool for cell and molecular biology research. Serious problems occur, however, when these techniques are applied to plant material. The presence of the cellulose wall can be a barrier to reproducible penetration of antibodies into cells and it often displays a confusing autofluorescence. A novel technique to prepare mitotic chromosome spreads from root tip meristems of germinating seeds is presented. Synchronous mitotic cells arrested in metaphase are converted into protoplasts using pectin and cellulose hydrolytic enzymes, and the purified protoplasts are fixed either in a methanol-acetic acid mixture to study DNA epitopes or in a nonextracting fixative to study chromosomal proteins. The latter fixative contains Triton X-100 to lyse the protoplasts and neutral formaldehyde to fix proteins by cross-linking. The protoplasts are immediately centrifuged onto microscopic slides as commonly done for mammalian cytogenetics. Using commercially available antibodies and both epifluorescence and confocal laser scanning microscopy, we demonstrated that the acid fixed chromosome slides are suitable for detection of DNA (anti-DNA antibody) or incorporated 5-bromodeoxyuridine (anti-BrdU antibody), while the cytospun formaldehyde and Triton X-100 fixed samples are convenient for detecting histones (antihistone antibody, pan). This technique should provide a general tool to study structural and functional domains of plant chromosomes.

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Section: Resultssupporting

confidence: 77%

A Cytospin Technique for Spreading Plant Metaphases Suitable for Immunofluorescence Studies

Hladilová

Široký

Vyskot

1998

Biotechnic & Histochemistry

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The pattern of histone H4 acetylation on the X chromosome during spermatogenesis of the desert locustSchistocerca gregaria

Wolf¹,

Turner²

1996

Genome

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We have used antibodies directed against histone H4 acetylated at lysine residue 5, 8, 12, or 16 and indirect immunofluorescence microscopy to probe chromosomes from spermatogonia and spermatocytes of the desert locust, Schistocerca gregaria. The autosomes showed bright overall fluorescence, indicative of high levels of H4 acetylation. In contrast, the X chromosome, which is facultatively heterochromatic during spermatogenesis of the locust, remained completely unstained in spermatogonia and secondary spermatocytes and showed only a small terminal fluorescent band in primary spermatocytes. This band probably corresponds to centromere associated constitutive heterochromatin. Thus, underacetylation is a cytogenetic marker for facultative heterochromatin, but not necessarily constitutive heterochromatin, during spermatogenesis of the locust. Scanning electron microscopy of chromosomes from prophase spermatogonia and prophase I spermatocytes revealed that underacetylation of histone H4 in the X chromosome was not accompanied by a chromatin organization visibly different from that of the autosomes. Transmission electron microscopy of mitotic spermatogonia showed that the X chromosome is separated from the autosomes in a small nuclear compartment of its own in prophase and telophase and associated with membranes in metaphase. In prophase I spermatocytes, autosomes and the sex univalent were in the same compartment. This compartmentalization may be responsible for the underacetylation and (or) transcriptional silencing of the X chromosome in spermatogonia mitosis.

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Preparation of Insect Chromosomes for Immunolabeling

Cited by 2 publications

References 37 publications

A Cytospin Technique for Spreading Plant Metaphases Suitable for Immunofluorescence Studies

A Cytospin Technique for Spreading Plant Metaphases Suitable for Immunofluorescence Studies

The pattern of histone H4 acetylation on the X chromosome during spermatogenesis of the desert locustSchistocerca gregaria

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