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1. Protocols for TEM specimen preparation
Part 1: Introduction
- The Light Microscope
- An explanation of how the light microscope works, how to use it, and how to get optimal results when using it.
- The Transmission Electron Microscope (TEM)
- An explanation of how the TEM works.
- EM visualization techniques
- An introduction to the routine methods used when preparing specimens for transmission electron microscopy.
Part 2: Methods
A. Negative staining
- Negative staining for transmission electron microscopy.
- Using coated grids.
- Using carbon films.
- Staining protocols.
- Staining solutions.
- Example of a negatively stained adenovirus.
B. Embedding in epoxy resins
- Epon, Araldite and mixtures
- Epon Embedding
- Spurr resin
- Introduction to Immunocytochemistry
- A brief overview of common available methods.
- Antibody labeling
- Labeling protocols using affinity markers.
- Embedding in resins for immunocytochemistry
- Describes the use of the specialized and not so specialized resins.
Lowicryls, LR White, LR Gold etc.
- Freeze substitution for embedding in resins
- A simple, inexpensive way of embedding in resin using freeze substitution methods.
- A detailed description of how to produce high quality cryosections of fixed,
cryoprotected biological material.
- Using resin sections for immunofluorescence
- A protocol for applying antibodies with fluorescent markers onto sections of resin-embedded material.
The thin sections help increase resolution.
- Preparation of colloidal gold probes
- See how easily, and inexpensively, these expensive probes can be prepared.
Excellent information if large volumes of gold probes are required.
- Silver enhancement of colloidal gold
- How to precipitate silver onto small colloidal gold particles
so that the signal can be detected by light microscopy.
- Morphology of Cryosections
- Cryosections do not have the same appearance, in electron micrographs, as conventionally embedded resin sections. Sometimes this different appearance is mistaken for poor morphology, sometimes the fine structure morphology is not good. Learn the difference and find out how to avoid poor morphology in these sections.
- Problems with Immunolabeling
- When labeling cells or sections with antibodies or other affinity markers, the results are not easily interpreted. Look here for some help.
E. General Protocols
- A semi-permenant mounting medium for immunofluorescence microscopy
- A recipe for Moviol,
an inexpensive mounting medium that is easy to prepare.
- How to coat metal specimen grids with Formvar
- Strong, thin support films are essential for routine TEM.
Here is a reproducable method for making thin films of formvar with details on how to mount them on metal specimen grids.
2. Protocols for light microscopy specimen preparation
- Immunolabeling for light microscopy
- Simple method for preparing thin sections of biological material for light microscopy. These sections give better high resolution information and provide better specimen preservation than even the thinnest cryostat section. May even be better than confocal optical sections!
- Mounting medium for immunofluorescence microscopy
- This mounting medium is easy to prepare, can be stored cold or frozen, can be used with anti-fade agents and will polymerize to give a dry, semi-permanent mount. If for some reason the sample is needed again, immesing in water will dissolve the medium again.
3. Further reference sources
- Reference books and journal articles
- List of books covering microscopy techniques and image interpretation.
- Links to microscopy-related sites
- Selected list of microscopy-related sites on the WWW.