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Preparation of Immunofluorescently Labeled Tissue Sections for Imaging at Low and High Magnifications in the Confocal
Jennifer Simpson1, Philippa C Hawes2
1Bioimaging, The Pirbright Institute, Pirbright, Woking, UK.
Abstract:
The confocal laser scanning microscope allows us to examine tissue sections in greater detail than a widefield fluorescence microscope. However, this requires samples to be better preserved than standard cryostat sections, which are not usually aldehyde-fixed. Thick sections (approximately 70 microm) of formaldehyde-fixed tissue can be cut using a vibrating microtome and subsequently labeled with primary and secondary fluorescent antibodies and/or fluorescent stains. When imaged in the confocal microscope, these samples allow us to collect high-resolution images, detailing the intracellular location of multiple proteins and structures. In this chapter, we describe the technique used to prepare vibrating microtome sections, using porcine tissue infected with African swine fever virus as an example. This technique can easily be applied to any animal tissue with any suitable combination of antibodies, depending on the hypothesis.
Insights
This study details a method for preparing thick, aldehyde-fixed vibrating microtome sections for high-resolution confocal microscopy. This technique enhances intracellular protein and structure visualization in animal tissues, exemplified by African swine fever virus research.
Area of Science:
- Microscopy
- Cell Biology
- Pathology
Background:
- Confocal laser scanning microscopy offers superior detail over widefield fluorescence microscopy.
- Standard cryostat sections lack aldehyde fixation, limiting preservation for high-resolution imaging.
- Enhanced sample preservation is crucial for detailed intracellular analysis.
Purpose of the Study:
- To describe a method for preparing thick vibrating microtome sections for confocal microscopy.
- To enable high-resolution imaging of intracellular components in aldehyde-fixed tissues.
- To provide a versatile technique applicable to various animal tissues and research hypotheses.
Main Methods:
- Tissue fixation with formaldehyde.
- Sectioning of fixed tissue using a vibrating microtome (approx. 70 µm thickness).
- Labeling with fluorescent antibodies and/or stains for immunofluorescence.
Main Results:
- High-resolution images detailing intracellular protein and structure localization were obtained.
- The technique successfully visualized viral infection in porcine tissue.
- Demonstrated the capability to image multiple proteins and structures simultaneously.
Conclusions:
- Vibrating microtome sectioning of formaldehyde-fixed tissue is a viable method for high-resolution confocal microscopy.
- This technique significantly improves the visualization of intracellular details compared to standard methods.
- The described protocol is adaptable for diverse research applications in animal pathology and cell biology.
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01:16Confocal Fluorescence Microscopy
01:12Immunofluorescence Microscopy
01:03Fixation and Sectioning
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...