There are 4 steps to sample preparation:
The processing of most samples begins with fixation to preserve morphology. A fixation method must be chosen that balances two characteristics:
'Good' preservation of cellular 3-D structure, and 'Adequate' access to antigenic sites.
The goal is to preserve sufficient cellular organization to allow identification of the features of interest, but not to destroy the antigenicity of the target. Fixation is also frequently combined with permeabilization to allow the staining solutions used in later steps access to the cytoplasm.
Commonly used histological methods of fixation and permeabilization often consist of treating the cells with solvents, such as alcohol or acetone. While these methods are quick acting precipitating fixatives, they are also good permeabilizing agents, but have one significant negative consequence: cellular shrinkage. The degree of shrinkage may be almost insignificant for monolayers of cells, but will distort tissue samples dramatically. To provide the ability to take full advantage of the three-dimensional reconstruction capability of the confocal microscope, the use of a fixative that does not destroy in vivo structure and organization must be found.
Glutaraldehyde (GA) is a commonly used fixative for electron microscopy, and does a terrific job of preserving microscopic structure. Unfortunately, glutaraldehyde has the unfortunate side effect of cross-linking many epitopes; this will render the tissue unlabelable. An additional undesirable side effect is the enhanced autofluorescence from residual unbound free aldehydes.
A good compromise is the use of paraformaldehyde (PFA) as a fixative. PFA will preserve most structures detectable at the confocal microscope level. Also, it will usually not obscure the epitopes of interest.
It is important to remember that different specimens may require a different fixation method. Testing and optimizing for each new sample type will insure that the best balance between preservation and labeling is obtained.
If a component of the cytoplasm or nucleus is to be labeled, the plasma membrane must be permeabilized. There are several ways to do this, and they depend on the fixation method chosen. Cells fixed with solvents do not require additional permeabilizing - as the solvent has already extracted enough of the membrane. Therefore solvent fixation is doubly efficient for this reason. Cells fixed with cross-linking aldehydes need to have the plasma membrane integrity breached by the use of chemical agents. Commonly used reagents include DMSO and detergents like Triton X-100, saponin or deoxycholate.
Permeabilization is another area, like fixation where fine tuning is necessary. Different detergents solubilize different molecules within the membrane, so it is necessary to know that the molecules you are interested in have not been washed away in the permeabilization step. The concentration of detergent is another detail that should be adjusted carefully. The idea is to selectively remove plasma membrane constituents to allow access to the cytoplasm without altering either the antigenicity or morphology of the sample.
Two basic techniques are used: direct labeling and indirect labeling.
Both labeling methods are suitable for confocal microscopy.
Direct labeling consists of using a fluorescently labeled primary antibody or chemical ligand to cause the structure of interest to become fluorescent. Advantages of this method include speed and ease of application. A potential disadvantage is lack of sensitivity (low signal intensity).
The indirect method involves binding a primary antibody to the epitope of interest, followed by a fluorescently labeled secondary antibody. The primary advantage of using this technique is the great amplification of signal possible through an antibody cascade. The disadvantages include increased complexity, the method is more time consuming, and there are often problems with non-specific antibody reactions.
Choosing a Fluorescent Label
The choice label depends upon the available equipment (lasers, filters) and the availability of certain fluorochromes conjugated to required antibodies for use in multiple labeling schemes. In general, the laser lines available dictate which fluorophores can be used. Recent advances in biochemistry have created new families of fluorophores with very favorable signal-to-noise and quantum efficiency (QE) properties. In particular, the Cy and Alexa dyes are particularly useful. Both families have high QEs, are very resistant to photobleaching, and are available in a variety of excitation/emission wavelengths.
Fluorescence detection is not the only way to use a confocal, however. The same light that passes through a specimen while imaging a fluorophore may be used to image the specimen by brightfield or phase-contrast or the Nomarski technique. Combining the emitted fluorescence and transmitted light signal is a particularly powerful technique for illustrating details of a cell layer that may not be fully fluorescent. An additional non-fluorescence based technique is reflection-mode confocal microscopy. Light reflected from the point of focus is collected and used as the source of signal for generating the image.
Fluorescently labeled cells and tissues exhibit a characteristic photobleaching curve in response to excitation by the laser. Much of the photobleaching can be attributed to the generation of free radicals. The use of free radical scavengers has been shown to decrease the rate of photobleaching. Common scavengers include n-propyl gallate, p-phenylenediamine and DABCO (1,4-diazobicyclo-(2,2,2)-octane). Live systems have been reported to reduced photobleaching in the presence of vitamin C or Trolox. If possible liquid embedding media should be used. Samples mounted in liquid media seem not to shrink in volume over time when sealed properly. Shrinking is at least less than in embedding media which harden by drying (like Mowiol, or ProLong from Molecular Probes). Samples embedded in liquid media like Vectashield or glycerol plus DABCO can be stored in the freezer (-20°C). In order to preserve the three-dimensional structure of the samples, a post can be constructed on the coverslip by brushing along each side of the slip with clear nail varnish prior to addition of the sample.
Most lab suppliers offer only 0.15 mm (nr. 1) cover slips, which are not adequate for high resolution microscopy. The objectives designed for the use with cover slips are generally designed for 0.17 mm cover slips. In order to preserve the three-dimensional structure of the samples, a post can be constructed on the coverslip by brushing along each side of the slip with clear nail varnish prior to addition of the sample.
Under certain conditions, some cover slips may show surface fluorescence generated by contaminating material. Clean your cover slips with analysis grade methanol, ethanol or acetone (any of these will probably work) and air-dry the cover slips prior to use.
Sealing the samples:
Nail polish or nail hardener or VALAP.
VALAP is a 1:1:1 mixture of vaseline, lanolin and paraffin. It is prepared by mixing the components in a bottle on a heating plate. The mixture is applied by thin tip and hardens immediately. It may be reheated and reused many times.