Cell Suspensions

De Waele et al. (17) recommended the immunolabeling of isolated cells while in suspension. After the final antibody incubation, cells are washed and cytocentrifuged and then silver enhanced and counterstained on the slide as usual. Although labeling may be stronger than in previously cytocentrifuged cells, the procedure involves centrifugation and resuspension of cell pellets after the labeling and washing stages, which may result in cell damage and depletion.

IGSS labeling has found more recent application in the fields of electron microscopy and deoxyribonucleic acid microarray technology (see Notes 12 and 13).

4. Notes

1. IGSS is still a fairly recent innovation in the fields of research and diagnostic histopathology, but the method is now making advances in some interesting applications (18). There have been reports describing labeling of leucocytes in hematology (19) and also in transplantation pathology (20) and cytology (21), the latter making use of Romanowsky counterstaining so that cells can be identified both morphologically and phenotypically at the same time. The IGSS method has been used in the monoclonal antibody diagnosis of B-cell lymphomas using paraffin-embedded material (22), and it has been suggested that cell surface antigens can be better demonstrated with periodate-lysine-paraformaldehyde-dichromate-fixed material (23). IGSS has been found to give superior results with a whole range of antisera used in routine paraffin histopathology, including regulatory peptides, intermediate filaments, and the calcium binding protein S100 (24). Using the procedures outlined in this chapter, it can be shown that IGSS offers a number of distinct advantages over other immunolabeling methods:

• gold sols are easy and cheap to produce

• gold particle size can be closely regulated; smaller size probes make a better nucleus for silver enhancement

• antibody/gold conjugation is relatively straight-forward and the resulting probes remain stable for a long time if refrigerated

• the method is economical as greater sensitivity permits incubation with more highly diluted antisera

• IGSS preparations are permanent and can be re-examined at later dates

• gold conjugates are non-hazardous and the procedure does not involve the use of carcinogenic chromogens. No precautions are required for handling or disposal of materials.

2. Problems with immunolabeling have been discussed in detail elsewhere (25,26) and are usually are attributed to one of the following: 1) Personal; has the operator made an error in the technique? 2) No antigen; the antigen may have been damaged during processing or there may be no antigen in the sample. 3) No antibody; the antibody may have been destroyed during preparation or there may be no specific antibody in the serum. Problems with IGSS labeling usually fall into two categories. Either there is no immunolabeling, or the labeling is so high that the accompanying background obscures the specific labeled sites. These problems are at first sight daunting but usually the remedy is quite simple. When there is no immuno-labeling, it is always advisable to run a positive control with each experiment to confirm that the reagents are functioning properly. If there is no immunolabeling on the experimental sections, the concentration of the antibody may be too low, or indeed too high. In the latter case, there is no room for the antibody to bind, therefore reducing the signal (25). It is possible that the antigen may be masked by the thickness of the section. Cut thinner sections or permeabilize the sections with a detergent, such as Triton X-100, or conduct proteolysis to unmask the antigen (26). A common problem is the fixation regime. If the fixation is too harsh, the antigenicity of the specimen will be lost, whereas if the fixation regimen is too light antigens and other material may leach from the tissue. Too much labeling is often accompanied by an unacceptably high background, and this may be caused by a very poor antibody. It is always wise to ascertain the titer and specificity of an antibody before immuno-labeling and estimate the expected results. If a suitable antibody has been chosen, the concentration of immunological reagents may be too high, or there may be a nonspecific attachment of reagents to the tissue. The former is remedied by conducting a series of dilutions of the reagents and silver development times and selecting concentrations that produce high signal to low noise. High background caused by the nonspecific attachment of reagents to the specimen is reduced by including 0.5% Triton X-100 in the buffers used for diluting the reagents. A preincubation of the specimen with 1% BSA and 1% gelatin in PBS (26) will block some of these nonspecific sites and will also, if necessary, block reactive electrostatic sites on the gold probes (27). Beltz and Burd (25) recommend that the addition of up to 0.05 M sodium chloride should reduce nonspecific labeling by preventing ionic interactions between the sera and the tissue. They warn also that high salt can interfere with low affinity antigen-antibody binding, and this technique should be used with care. Beltz and Burd (25) also recommend that if there is trouble with background immunolabeling, the antiserum can be cross adsorbed with fresh tissue from the same host that does not contain the antigen in question. If these fail, they suggest the selection of another antibody against the same antigen. Although immunocytochemistry

Bacteria Temperature Optimum Curve
Fig. 3. Temperature-time curve for standardizing silver enhancement. Silver enhancement time in IGSS increases with lower operating temperature. The use of a calibration curve assists in optimizing the enhancement procedure.

is now a routine technique, care must still be taken to obtain optimum results. Pay particular attention to freshness of reagents, especially buffers, which will become contaminated with bacteria that may interfere with immunolabeling. During incubations, care should be taken to prevent the specimens from drying as evaporation will concentrate the antibody solutions leading to increased background deposition.

3. Problems with nonspecific background silver deposition: background silver deposition may result from the use of poor-quality antibodies, incorrectly diluted antibodies, old silver-enhancing solutions, poor-quality distilled water, or incorrect silver enhancement times. The silver enhancement procedure is temperature dependent and where laboratory temperatures vary a lot, especially at different times of the year, it is useful to construct a standardized temperature-enhancement time graph and keep it readily available at the bench (Fig. 3). As an example, adequate silver enhancement may take only 5 min at 25 °C whereas the same result may take up to 15 min to obtain at 15°C. Enhancement times may be controlled more precisely by storing the enhancer components in the refrigerator at

Table 3

Historical Development of Specialist Microscopy Procedures for Viewing IGSS

Authors/dates

De Waele (1986) (31) Kazama et al. (1994) (32) Uriel et al. (1995) (33) Neri et al. (1997) (34)

Technique

Reflection contrast microscopy Viewing of living cells attached to slides Epipolarization of immunogold alone

Epipolarization of IGSS Confocal microscopy of IGSS. Use of reflection mode especially in double labeling.

4°C. Although autonucleation should not be a problem when using modern commercial enhancement kits, silver deposition may be avoided by performing the reaction in a darkroom with a Safelight 5902 or F904.

4. Epipolarization Microscopy: an interesting extension of the IGSS techniques lies with some of the optical properties of silver grains. Sites of silver deposition are strikingly demonstrated under dark ground illumination, although it is not possible to see the surrounding tissue morphology at the same time. Most fluorescence microscopes may now be adapted to permit the study of IGSS under epipolarization, a procedure that has evolved from reflection contrast microscopy (see Table 3). Powerful episcopic illumination from a mercury vapor light source passes through interchangeable filters, an adjustable diaphragm and into the special epipolarization block. This block contains a ultraviolet protection filter, a dichroic half-mirror, and an analyzer. The only light that can pass through the polarizer and return from the sample to the analyzer is the intense, back-scattered light from silver labeling (ref. 35; Fig. 4). The epipolarized light appears as a bright turquoise signal. The image is usually strong enough to allow ordinary diascopic light to be used at the same time for visualizing other tissue structures. Epipolarization is especially useful when silver enhancement is faint, in automated image analysis where the most clear, precise labeling is desirable and also when a heavy counterstain has been used and the silver deposit is unclear. The use of water or oil, medium-power, objective lenses is recommended because problems with glare may be encountered at low power magnification when a dry objective is used. Also, as epipolarization microscopy greatly increases the sensitivity of IGSS detection, any background silver deposition will also be illuminated more sharply. Epipolarization will often still give a clear result with IGSS preparations that have either faded or turned brown after incomplete stabilization with sodium thiosulfate.

5. Two aspects of IGSS make the technique highly suitable for subsequent study using image analysis. Firstly, the intense, sharp, black reaction product is easily discriminated by either monochrome or full-color image analysis systems. Second, epipolarization may be used to provide even more discrimination between the IGSS signal and the background, which is particularly useful when a heavy coun-terstain has been employed for identifying other features of the preparation. A procedure has been described (36) whereby lymphocyte subpopulations labeled with IGSS may be enumerated by examining the total, hematoxylin-counterstained cell population under diascopic white light and then changing to epipolarizing illumination to count the proportion of IGSS-labeled cells that are present (Fig. 4). Occasionally, caution is required with the interpretation of computer-analyzed IGSS data, for example, when other black tissue components are present such as carbon particles within lung sections (37).

6. Whereas most immunohistochemical procedures may only successfully use a delicate nuclear counterstain, a wide variety of counter-stains may be used after IGSS labeling, including trichromes and more selective techniques such as those used in the identification of micro-organisms. The Romanowsky procedure has been used as a counterstain for the morphological examination of lymphocyte populations previously labeled with IGSS incorporating lymphocyte subset markers (21,36). Only silver impregnation procedures and

Fig. 4. (continued on opposite page) Flow diagram illustrating the application of image analysis to IGSS using a conventional diascopic white light -1 episcopic polarized light double-illumination light microscope system. Adapted from ref. 35 with permission.

techniques giving a black or near-black coloration should be avoided; epipolarization may be helpful in the latter situation but silver impregnation preparations behave variably under epipolarized light.

7. The color development of silver grains in IGSS has been used to convert the black silver signal to red, yellow, or blue-green (38). The method is a histochemical application of the chemical reactions more traditionally used in color photography.

8. The combination of IGSS with a variety of different immunoenzyme and immunofluorescence procedures has been evaluated (39), and the application of IGSS followed by immunoalkaline phosphatase seems to provide the most successful results.

9. IGSS is now being used for locating DNA probes with in situ hybridization, especially incorporating the new 1-nm gold labels (40).

10. Work has shown that IGSS labeling procedures can be performed more rapidly using microwave stimulation (41).

11. IGSS may also be performed on cryostat sections, which may then be plastic embedded for semithin sectioning (42).

12. Although the IGSS technique was originally introduced for use in light microscopy, the procedure has also found application in combined light microscope and electron microscope studies (43) as well as in the electron microscope field alone (44).

13. The use of silver enhancement of colloidal gold is not restricted to the microscopical study of cells. The development of microarrays has revolutionized gene expression analysis and molecular diagnosis through miniaturization and the multiparametric features. Microarrays have traditionally been detected in fluorescence, but recently a method using nanogold particles has been developed. The signal generates results from the precipitation of silver onto nanogold particles bound to streptavidin, the latter being used for detecting biotyinylated DNA (45).

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