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A method for using low-temperature embedding media for electron microscopy of cells grown on microporous supports
V L Rudick1, A M Brun-Zinkernagel, M J Rudick
1Department of Anatomy and Cell Biology, Texas College of Osteopathic Medicine, Fort Worth 76107.
Abstract:
Attempts were made to embed Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose microporous supports in Lowicryl, a medium designed for the retention of antigenicity for electron microscopic immunocytochemical studies. It was found that the membrane fragmented during the prescribed embedding procedure leading to loss of the cell sample. This necessitated the formulation of a new combination of fixative and dehydration agents which would allow: (i) preservation of membrane and cellular integrity; (ii) infiltration and embedding in Lowicryl; and (iii) detection of a specific antigen in thin sections. The cellular monolayer and organelle profiles were best preserved with glutaraldehyde fixation at room temperature followed by ethanol dehydration. Since the latter was carried out at temperatures attainable with an ice-salt bath, there was no need for a special ultralow-temperature apparatus. This procedure was applied to a MDCK cell clone that consisted of stable secretors of human growth hormone (hGH), as a result of being transfected with a plasmid containing the hGH gene. Thus, it was demonstrated that hGH could be detected by immunogold labeling of thin sections and localized to specific cellular structures. The procedure developed in this report is applicable to cells grown on two other supports and may be extended further.
Insights
Researchers developed a new method for embedding Madin-Darby canine kidney (MDCK) cells for electron microscopy. This technique preserves cell integrity and allows for antigen detection, improving immunocytochemical studies.
Area of Science:
- Cell Biology
- Microscopy
- Immunocytochemistry
Background:
- Standard embedding methods for electron microscopy often compromise cell integrity.
- Lowicryl embedding is ideal for antigen retention but can damage cell structures.
- Madin-Darby canine kidney (MDCK) cells are a common model for cell biology research.
Purpose of the Study:
- To develop a novel embedding protocol for MDCK cells that preserves cellular integrity and allows for antigen detection.
- To optimize fixative and dehydration agents for Lowicryl embedding in electron microscopy.
- To validate the new method using human growth hormone (hGH) detection in transfected MDCK cells.
Main Methods:
- Cells were fixed with glutaraldehyde at room temperature and dehydrated with ethanol at ice-salt bath temperatures.
- Optimized fixative and dehydration agents were used for infiltration and embedding in Lowicryl.
- Immunogold labeling was employed to detect specific antigens in thin sections of embedded cells.
Main Results:
- The new protocol successfully preserved the monolayer and organelle profiles of MDCK cells.
- Cellular and membrane integrity were maintained throughout the embedding process.
- Human growth hormone (hGH) was successfully detected and localized to specific cellular structures using immunogold labeling.
Conclusions:
- A robust embedding method was established for electron microscopic immunocytochemistry of MDCK cells.
- The developed procedure enhances antigen detection by preserving cellular structures.
- This technique is adaptable for various cell types and supports, broadening its applicability in cell biology research.