This is a cached page for the URL (http://www.hei.org/research/depts/aemi/tec.htm). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Advanced Electron Microscopy & Imaging
House Ear InstituteHomeContact UsDirectorySearchSite Map
Research
Departments
General Overview
Scientific Projects
Departments
Scientists
   
 

These pages contain information on light microscopy,
transmission electron microcopes, cryosectioning,
immunocytochemistry and other imaging
techniques used in modern
biomedical research.

Contents

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.
Introduction.
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

C. Immunocytochemistry

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.
Cryosectioning
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.

D. Troubleshooting

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.

 

 
 

Updated February 15, 2001
House Ear Institute