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Fixation and Sectioning01:03

Fixation and Sectioning

Two basic types of preparation are used to visualize specimens with a light microscope: wet mounts and fixed specimens.
The simplest type of preparation is the wet mount, in which the specimen is placed in a drop of liquid on the slide. A liquid specimen can be directly deposited on the slide using a dropper. Solid specimens, such as skin scraping, can be placed on the slide before adding a drop of liquid to prepare the wet mount. Sometimes the liquid is simply water, but stains are often added...
Euchromatin01:01

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The extent of chromatin compaction can be studied by staining chromatin using specific DNA binding dyes. Under the microscope, the dense-compacted regions take up more dye, appearing darker, while the less-compact areas take up less dye and appear lighter. Based on the compaction level, chromatins are classified into two primary forms – euchromatin and heterochromatin.
Euchromatin is the less dense region of the chromatin and stains lighter. Euchromatin contains histone H3 extensively...
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Chromatin immunoprecipitation, or ChIP, is an antibody-based technique used to identify sites on DNA that bind to transcription factors of interest or histone proteins. It also helps determine the type of histone modifications such as acetylation, phosphorylation, or methylation.
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Epigenetics is the study of inherited changes in a cell's phenotype without changing the DNA sequences. It provides a form of memory for the differential gene expression pattern to maintain cell lineage, position-effect variegation, dosage compensation, and maintenance of chromatin structures such as telomeres and centromeres. For example, the structure and location of the centromere on chromosomes are epigenetically inherited. Its functionality is not dictated or ensured by the underlying DNA...
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The chromatin structure, especially...
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The extent of chromatin compaction can be studied by staining chromatin using specific DNA binding dyes. Under the microscope, the dense-compacted regions that take up more dye are called heterochromatin. Heterochromatin is further classified into two forms – constitutive heterochromatin and facultative heterochromatin.
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Changes in chromatin structure during processing of wax-embedded tissue sections.

Elizabeth Kerr1, Tomoharu Kiyuna, Shelagh Boyle

  • 1Breakthrough Breast Cancer Research Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK.

Chromosome Research : an International Journal on the Molecular, Supramolecular and Evolutionary Aspects of Chromosome Biology
|July 28, 2010
PubMed
Summary
This summary is machine-generated.

Investigating nuclear organization using immunofluorescence (IF) and fluorescence in situ hybridisation (FISH) in fixed tissues reveals that processing steps significantly alter nuclear architecture and chromatin texture.

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genomics

Background:

  • Immunofluorescence (IF) and fluorescence in situ hybridisation (FISH) are key techniques for studying nuclear and chromatin organization.
  • Current research emphasizes the need to study nuclear organization in situ within native tissue architecture, moving beyond cultured cells.
  • Formalin-fixed paraffin-embedded (FFPE) tissues are crucial for analyzing archival clinical samples but require additional processing for IF and FISH.

Purpose of the Study:

  • To quantify structural changes in the nucleus and chromatin caused by processing steps required for IF and FISH on FFPE tissues.
  • To assess the impact of specific treatments, such as microwave antigen retrieval, on nuclear architecture and chromatin texture.
  • To inform the interpretation of nuclear organization data derived from FFPE tissue sections.

Main Methods:

  • Analysis of nuclear and chromatin structure in FFPE tissue sections subjected to various processing treatments.
  • Quantification of structural alterations using microscopy and potentially image analysis techniques.
  • Comparison of structural features before and after specific retrieval methods, including microwaving.

Main Results:

  • Processing steps, particularly microwaving for fixation reversal, significantly alter nuclear architecture.
  • Chromatin texture undergoes notable changes due to the applied treatments.
  • These alterations impact the fidelity of nuclear organization studies using FFPE samples.

Conclusions:

  • The processing of FFPE tissues for IF and FISH introduces significant changes to nuclear and chromatin structure.
  • Researchers must carefully consider these induced alterations when interpreting nuclear organization data from FFPE samples.
  • Understanding these modifications is critical for accurate analysis of nuclear organization in native tissue contexts.