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Materials for Horseradish Peroxidase-Conjugated Second Antibodies
1. 0.05M Tris buffer pH 7.6
2. DAB tablets (Sigma catalogue No. 5905)
3. Hydrogen peroxide 30% (stored at 4jaC for up to 1 month)
4. Whatman No. 1 filter paper
1. Dissolve one DAB tablet in 15 ml Tris buffer (0.05 M, pH 7.6).
2. Add 0.1 ml of a 3% solution of hydrogen peroxide. Filter the precipitate if necessary.
3. Add solution to the well sufficient to cover the cells.
4. Incubate at room temperature for up to 20 min. Stop the reaction by washing with water.
5. Mount with 50% glycerol or Permount.
Materials for Alkaline Phosphatase-Conjugated Second Antibodies
1. Naphthol AS-Bl-phosphate
2. New fuchsin (Sigma catalogue no. N0638)
3. Sodium nitrate
4. Naphthol AS-TR (Sigma catalogue no. N5875)
5. 0.2 M Tris buffer (pH 9.6)
6. 2 N HCl
8. 20 mM EDTA
1. Dissolve 1 mg of new fuchsin in 0.25 ml 2N HCl.
2. Dissolve 1 mg sodium nitrate in 0.25 ml H2O.
3. Dissolve 10 mg naphthol AS-TR in 0.2 ml Dimethylformamide.
4. Add the new fuchsin solution to the sodium nitrate solution and mix for 1 min. Add this to 40 ml of 0.2 M Tris (pH 9.6) buffer.
5. Add naphthol AS-TR solution to the solution in step 4.
6. Cover the well with the solution and leave for up to 30 min at ambient temperature. Stop the reaction with 20 mM EDTA.
7. Mount with 50% glycerol or Permount.
Generally, bright-field microscopy is used in the observation of specimens that are fixed and stained, such as histology sections. The microscope used is usually upright as opposed to inverted. This type of conventional illumination will not reveal brightness differences between the structural details in the living cell and its surroundings because the image lacks contrast, but it is ideally suited for pathology and histological applications in which the morphology and immunohistochemical staining are of paramount importance in preserved specimens. Bright-field microscopy is of little use for viewing living tissue culture plates, but it may prove useful to have a second bright-field microscope if the work in the laboratory depends a lot on stained tissue or cultures or uses immunohistochemical staining to gather data.
In dark-field microscopy, the illuminated object is bright against a dark background. This is achieved by means of a central solid spot in the condenser that allows illumination of the cell or particle only from the side. The extremely high contrast provided by this type of illumination makes it possible to visualize very small particles in the cell that are beyond the resolution of the phase contrast microscope.
Dark-field microscopy is a method that is useful when trying to detect minute particles. Examples might include autoradiography or in situ hybridization in tissue sections or cultures grown on slides and processed. The DIC condenser used for dark-field illumination creates a cone of light in which the direct or oblique rays do not enter the objective. When these rays intercept a particle, however, they are scattered into the objective, causing the particle to appear luminescent against a black background (Fig.
6.11). Dark field can best be viewed with the smaller magnification objectives, that is, 8x or 10x. A 20x objective can be used, but it is preferable to use one that does not have high light gathering capability (one with a low NA), to avoid an undesirable high background. Special objectives for dark-field use are available if higher magnifications are needed.
A dark-field format can be achieved through the use of a phase contrast condenser and bright-field (nonphase) objectives. The phase ring in the condenser should be larger than the objective used. This will then produce a dark-field cone of light where the direct ray of illumination will not enter the objective. Usually, the use of a 10x objective and a ph3 annular ring will be sufficient. There are also after-market sources for obliquely lit slide holders, which most popular microscope stages can accommodate, that create a dark-field effect while allowing the additional use of filters on the microscope itself for further image enhancement.
A frozen section of rat testis treated with autoradiography after [125I] inhibin has been bound, and then the section was counterstained. The left panel shows a bright-field view that allows one to see cellular morphology, while the dark-field view of the same section on the right emphasizes the silver grains.
Adding the Third Dimension
A vexing problem in conventional microscopy has been that objectives with a numerical aperture high enough to resolve fine structure will be limited by an extremely shallow depth of field. This restricts the information gathered to two dimensions in which the complex structural relationship of the subcellular components is difficult, if not impossible, to discern. Even DIC illumination, which provides a surface view, is two-dimensional and suffers from a shallow depth of field. Microscopes that allow threedimensional viewing of specimens on slides are relatively new. Scanning confocal microscopes utilize a computerized reconstruction of data viewed through sections along the z-axis, thereby generating a threedimensional view (Boyde, 1985, 1986). The Edge high-definition stereo light microscope (Edge Scientific Instrument Co., Santa Monica, CA), as seen in Fig. 6.12, is set up to allow real-time threedimensional viewing in phase, bright field, dark field, or fluorescence. Its illumination system supplies distinct left and right images for true stereoscopic observation as well as three-dimensional and conventional photomicroscopy. As data acquisition and analysis software become available, this