A protocol for preparation of chromosome spread and processing for transmission electron microscopy

Authors

  • Tatsuo Oguro Division of Pathology, Chiba Hokuso Hospital, Nippon Medical School, Chiba, Japan.
  • Mohammad Ghazizadeh Central Institute for Electron Microscopic Researches, Nippon Medical School, Tokyo, Japan.

Keywords:

Method, flat embedding, transmission electron microscopy, human chromosome, ultrastructure

Abstract

Transmission electron microscopic (TEM) observation of human chromosome ultrastructure may provide useful information on various abnormalities. Due to the difficulties in detaching metaphase chromosome spreads from the glass slide, this approach has been restricted. We introduce a simplified protocol in which the metaphase chromosome spreads are made on a flexible thermoplastic membrane (Aclar film) glued to the routine microscopic glass slides. The chromosome spreads are then impregnated with 1% osmium tetroxide, stained with freshly prepared 2% tannic acid, dehydrated, and flat-embedded in epoxy resin. After polymerization, the epoxy resin sheet is easily detached from the Aclar film and all chromosome spreads retain on the resin sheet. Each chromosome spread is identified under a light microscope, demarcated, trimmed then attached to a pre-polymerized blank epoxy resin block. Ultrathin sections are cut and observed under a transmission electron microscope. Application of this method to chromosome research is suggested to provide useful information on the chromosome morphology and ultrastructure in relation to various conditions and/or function.

Author Biographies

Tatsuo Oguro, Division of Pathology, Chiba Hokuso Hospital, Nippon Medical School, Chiba, Japan.

Laboratory Chief, Division of Pathology, Chiba Hokuso Hospital, Nippon Medical School, Chiba, Japan.

Mohammad Ghazizadeh, Central Institute for Electron Microscopic Researches, Nippon Medical School, Tokyo, Japan.

Central Institute for Electron Microscopic Researches, Nippon Medical School, Tokyo, Japan.

References

Squarzoni S, Cinti C, Santi S, Valmori A, Maraldi NM. Preparation of chromosome

spreads for electron (TEM, SEM, STEM), high and confocal microscopy. Chromosoma 103:381-392, 1994.

Wen GY, Jenkins EC, Goldberg EM, Genovese M, Brown WT, Wisniewski HM. Ultrastructure of the fragile X chromosome: New observations on the fragile site. Am J Med Genet 83:331-333, 1999.

Ghazizadeh M, Sasaki Y, Oguro T, Sato S, Egawa S, Inoue K, Shimizu H, Kawanami O. A novel technique for observing the internal ultrastructure of human chromosomes with known karyotype. Microsc Microanal 14:357-361, 2008.

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Published

2015-04-15

Issue

Vol. 2 No. 2 (2015)

Section

Articles

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