TECHNICAL NOTE |
Correspondence to: Nobuteru Usuda, Dept. of Anatomy and Cell Biology, Shinshu Univ. School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan.
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Summary |
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The protein Agold technique has been widely applied for visual localization and quantification of various antigens by electron microscopy. Observation of specimens stained by the protein Agold technique with conventional light microscopy is difficult because of insufficient sensitivity of the staining. Light microscopic visualization and quantification of the reaction products were attempted employing a confocal laser scanning microscope (CLSM). Liver tissues of normal and peroxisome proliferator-treated rats were fixed and embedded in Lowicryl K4M resin. Ultrathin and thin sections were stained for catalase and a peroxisome-specific ß-oxidation enzyme by the protein Agold technique. Ultrathin sections were observed by electron microscopy and the labeling density for each enzyme was analyzed with an image analyzer. Thin sections were observed with a CLSM in the reflection mode and the intensity of the light reflection was analyzed under the same conditions for all specimens. A comparison of these two observation procedures was also attempted using liver tissues stained with various concentrations of the antibody for catalase. The intensity of the reflection for each, as observed by CLSM, correlated well with the labeling density observed by electron microscopy. CLSM made it possible to quantify and to directly observe protein Agold staining at the light microscopic level. (J Histochem Cytochem 47:13431349, 1999)
Key Words: confocal laser scanning, microscopy, protein Agold technique, peroxisome, liver, quantification
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Introduction |
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THE PROTEIN A–GOLD TECHNIQUE has been widely used to visualize the localization of various antigens by electron microscopy, because of its high sensitivity and high image resolution (
Confocal laser scanning microscopy (CLSM) is coming to be commonly used in the observation of various kinds of specimens, most commonly with fluorescence, and quantification of the fluorescent reaction products has also been attempted (
In this study, hepatic peroxisomes were used as the subject of observation by CLSM in the reflection mode. The peroxisome is a spherical cell organelle with a diameter of about 0.51.0 µm (
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Materials and Methods |
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Tissue Preparation
Liver tissues of 14-week-old male Wistar rats, fed normal or 1% DOP-containing diets (
Immunoelectron Microscopy
Staining was performed as previously described (
Quantification of Gold Particles on Electron Micrographs
The labeling density, i.e., the number of gold particles per unit area of peroxisomes, was estimated for each enzyme in hepatocytes in the pericentral area of hepatic lobules. The area of peroxisomes, the cytoplasm, and the number of gold particles on them were measured using the computer software Ultimage (Graftek; Voisins le Bretonneux, France). Fifty peroxisomes photomicrographed at a magnification of x20,000 were evaluated in each group. The total labeling index was calculated as volume density x labeling density for electron microscopic observation (
Confocal Laser Scanning Microscopy and Quantitative Measurement
One-µm sections were collected on aminopropyl-triethoxysilene-coated glass slides. They were stained in the same solution and with the same incubation time as for immunoelectron microscopy. Specimens were examined with a confocal laser scanning microscope (TCS NT; Leica Lasertechnik, Heidelberg, Germany). The CLSM was equipped with an ArKr laser having three major emission lines at wavelengths of 488 nm, 568 nm, and 647 nm. The 488-nm line was employed because the intensity of this line is the most sustainable. The reflecting light from the specimens at 488 nm was captured by a photomultiplier. The reflection light incidents of the images were digitized in 256 grades (28 grades), i.e., the reflection from the area of sinusoids was set to 0 and that from normal peroxisomes stained for catalase to 255, so that the values of all four groups fitted this range. Fifteen randomly selected lines under which 50 peroxisomes were present were evaluated in each group. The value for each peroxisome was obtained by subtracting the average of the cytoplasmic reflection from the peroxisomal reflection. Figure 1 shows an example of the procedure for measurement of the light reflection from a specimen stained by the protein Agold technique. Figure 1A is a digitized image of normal liver tissue stained for catalase. The sinusoid, cytoplasm, and three intense signals are located under the line. Figure 1B shows the line intensity of the reflection light measured. The same measurement condition as in normal hepatocytes stained for catalase was applied to the other three kinds of specimens. Control staining was performed using the IgG fraction of nonimmune serum instead of antibodies.
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Effect of Antibody Concentration
Another experiment was carried out to determine the effect of the concentration of the antibody on the relationship between the data of the two procedures: measurement with electron microscopy and that with CLSM. Serial sections of DOP-treated liver tissue were stained for both procedures as described above with various concentrations of anti-catalase antibody, 0.32, 1.6, 8, and 40 µg/ml of IgG.
Statistical Analysis
Data obtained by immunoelectron microscopy and by CLSM were statistically analyzed by analysis of variance (ANOVA) and the differences between respective groups were judged to be significant at p<0.05.
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Results and Discussion |
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In the observations using CLSM, an increase in the number of peroxisomes was evident in the DOP-treated hepatocytes compared with normal hepatocytes (Figure 2). In addition, the difference in the intensity of the reflection from peroxisomes could be recognized in the four groups by careful observation. A small number of intense light reflections were observed over normal hepatocytes stained for catalase (Figure 2A), and many but less intense reflections were observed over DOP-treated hepatocytes (Figure 2B). A small number of light reflections were observed over normal hepatocytes stained for PH (Figure 2C). However, the observation was difficult because of the weakness of light reflections. Many intense light reflections were observed over DOP-treated hepatocytes (Figure 2D). On electron microscopic observation, peroxisomes of normal hepatocytes stained for catalase by the protein Agold technique were densely labeled with gold particles (Figure 3A) but those of DOP-treated hepatocytes were less so (Figure 3B). Staining of peroxisomes of normal hepatocytes for PH was rather weak (Figure 3C) but those of DOP-treated hepatocytes were densely labeled (Figure 3D).
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The labeling density for peroxisomes calculated on electron micrographs and the intensity of light reflection from peroxisomes measured by CLSM are summarized in Figure 4. The intensity levels of light reflections measured by CLSM for the four were in the following order: normal peroxisomes stained for catalase; DOP-treated peroxisomes stained for PH; DOP-treated peroxisomes stained for catalase; and normal peroxisomes stained for PH (Figure 4A). The labeling densities for the four groups calculated on electron micrographs were in the same order (Figure 4B). The labeling density on electron microscopic observation and the intensity of light reflection by CLSM were in a good mutual relationship, approximately in a line (Figure 4C). A similar comparison of the data from these two procedures was made on another measurement employing serial sections of DOP-treated hepatocytes stained for catalase with various concentrations of the antibody. The labeling density and the intensity of the light reflection were ordered according to the concentration of the antibody. These two were also well correlated with high concentrations of the antibody, but the value for the lowest (0.32 µg/ml) appeared not to be within the linear range (Figure 5).
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As a way of estimating the changes in the total amount of antigens on electron microscopic specimens stained by the protein Agold technique, the total labeling index or immunolabeled concentration (
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There are several representative immunoelectron microscopic techniques. The immunogold techniques include the protein Agold technique, the ferritin technique, and the immunoperoxidase technique. The immunogold technique has various advantages because of the particulate and electron-dense reaction products of colloidal gold particles; the ultrastructure behind the gold particles is clearly visible and a higher resolution of the picture is obtainable than with other techniques. Another advantage is quantification of the particles for estimating the amount of antigen in the fine structure, i.e., the number or density of gold particles correlates with the amount of antigen (
Although the immunogold technique has ordinarily been used for electron microscopy, it can also be used for light microscopic observation without any intensification, thanks to the CLSM (
In the observation of specimens stained by the protein Agold technique, the change in the labeling density of gold particles shown by electron microscopic observation correlates with the change in the reflection intensity measured by CLSM observation, which is especially suitable for specimens densely labeled by protein Agold particles. Because a wider area of specimens can be observed by CLSM than by electron microscopy, a highly reliable estimate of the amount of antigens in the tissues stained by the protein Agold technique is possible by CLSM observation combined with electron microscopy.
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Acknowledgments |
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Supported in part by grants-in-aid for scientific research from the Ministry of Education of Japan.
We gratefully thank Dr T. Hashimoto (Department of Pathology, Northwestern University Medical School) for kind guidance in purifying enzymes and raising antibodies, and Y. Furihata (Kamijo Kikai Co., Ltd.) for technical advice.
Received for publication November 23, 1998; accepted April 27, 1999.
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