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Perceiving mitosis in eukaryotic cells
Abstract:
A sensitive method has been developed for visualizing eukaryotic cells in mitosis (M) phase. It employs Zenker's fixative, which makes the plasma membrane but not the nuclear envelope permeable to immunoglobulins. Zenker's-fixed cells are exposed to an antibody which recognizes a major constituent of chromatin. In this case the antibody is a monoclonal (MC 21) which recognizes histone H2b. Because cells in M phase do not have an intact nuclear envelope, the antibody has access to and interacts with their chromatin. The presence of a nuclear envelope in Zenker's-fixed interphase cells precludes access of the antibody to the nuclear chromatin. Consequently, this indirect immunofluorescence procedure selectively labels M-phase cells. At high enough magnification some details of the chromatin figures are revealed. MC 21 recognizes the chromatin of cells of many different species. With appropriate fixation it can be used effectively on cells in culture. With some procedural modifications it can also be used with more complex tissue systems. Detailed mitotic patterns for chick embryos up to Day 3 of development have been obtained by this method.
Insights
A new method uses Zenker's fixative and a histone H2b antibody to selectively label cells in mitosis (M) phase. This technique allows for detailed visualization of mitotic patterns in various species and tissues.
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- Visualizing specific cell cycle phases is crucial for understanding cell division.
- Traditional methods for identifying mitotic cells can be complex or lack specificity.
Purpose of the Study:
- To develop a sensitive and selective method for identifying eukaryotic cells in mitosis (M) phase.
- To utilize indirect immunofluorescence for visualizing mitotic chromatin.
Main Methods:
- Employing Zenker's fixative to selectively permeabilize the plasma membrane while preserving nuclear envelope integrity in interphase cells.
- Using a monoclonal antibody (MC 21) targeting histone H2b, a major chromatin constituent.
- Applying indirect immunofluorescence to detect antibody binding to chromatin in M-phase cells where the nuclear envelope is absent.
Main Results:
- The method selectively labels M-phase cells due to antibody access to chromatin only when the nuclear envelope is disrupted.
- Details of chromatin figures within M-phase cells are observable at high magnification.
- The monoclonal antibody MC 21 demonstrates cross-reactivity with chromatin across various species.
- The technique is effective for cells in culture and adaptable for complex tissue systems, including chick embryos.
Conclusions:
- This indirect immunofluorescence method provides a sensitive and selective approach for visualizing eukaryotic cells in mitosis.
- The technique allows for detailed analysis of mitotic patterns and chromatin structures.
- The method's versatility makes it applicable to diverse biological systems for cell cycle research.