ARTICLE |
Correspondence to: Manoj Raje, Inst. of Microbial Technology, Sector 39A, Chandigarh 160114, India. E.mail: manoj@imtech.ernet.in
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Summary |
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We developed an ELISA-based method for rapid optimization of various tissue processing parameters in immunogold labeling for electron microscopy. The effects of aldehyde fixation, tannic acid, postfixation, dehydration, temperature, and antigen retrieval on antibody binding activity of Vitreoscilla hemoglobin (VHb) expressed in E. coli cells were assayed by ELISA and the results confirmed by quantitative immunogold labeling transmission electron microscopy (TEM). Our results demonstrated that low concentrations (0.2%) of glutaraldehyde fixation caused minimal loss in total binding compared to higher concentrations. Dehydration in up to 70% ethanol resulted in some distortion of cellular ultrastructure but better antibody binding activity compared to dehydration up to 100%. Postfixation or incorporation of tannic acid in the primary fixative caused almost total loss of activity, whereas antigen retrieval of osmium-postfixed material resulted in approximately 90100% recovery. The sensitivity of detection of proteins by immunogold labeling electron microscopy depends on the retention of antibody binding activity during tissue processing steps, e.g., fixation and dehydration. Our study indicated that an ELISA-based screening method of various tissue processing procedures could help in rapid selection and optimization of a suitable protocol for immunogold localization and quantification of antigen by TEM. (J Histochem Cytochem 49:355367, 2001)
Key Words: antigen detection, quantitation, transmission electron microscopy, ELISA, tissue processing, antigen retrieval, artificial immunospecimen, immunogold labeling
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Introduction |
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CHOICE OF TISSUE PROCESSING STEPS is crucial for efficient detection and quantification of target antigen in cells by immunogold labeling electron microscopy. Many protocols involve treatment with chemical crosslinking agents, subsequent exposure to various gradients of alcohol for dehydration, and exposure to temperatures up to 60C for polymerization. All of these steps, although attempting to preserve sample ultrastructure, also affect the native antibody binding activity of the antigen under study (
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Materials and Methods |
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VHb Antigen and Anti-VHb Antibody
E. coli DH5 cells harboring the plasmid PUC8:15 expressing VHb protein (
ELISA of VHb Antigen
Fifty µl of lysate containing 2.5 µg of protein (
Fixation Treatment
Fifty µl of different fixatives, constituted in either PBS or 0.1 M sodium cacodylate buffer (CB), was added to each well and allowed to act for 30 min at 4C. The different fixatives evaluated were as follows: (a) 0.2% glutaraldehyde (EM Sciences; Ft Washington, PA) + 4% paraformaldehyde (Polysciences; Warrington, PA); (b) 0.5% glutaraldehyde + 4% paraformaldehyde; (c) 1% glutaraldehyde + 4% paraformaldehyde; (d) 2.5% glutaraldehyde + 2.5% paraformaldehyde; (e) 0.2% glutaraldehyde + 4% paraformaldehyde supplemented with 0.1, 0.2, 0.5, 1, or 2% tannic acid (s.d. Fine-Chem Ltd; Boisar, India); (f) 0.2% glutaraldehyde + 4% paraformaldehyde followed by 1% OsO4 (EM Sciences). Wells to which OSO4 was added were sealed with parafilm.
Treatment with Alcohol
Coated antigen in each well was exposed to either 30%, 70%, or 100% ethanol for 30 min at 4C before being assayed for binding to antibody.
Heat Treatment
A set of eight wells was exposed to 37C or 60C for 24 hr before being processed for ELISA.
Simulation of Cell Processing with an Artificial Immunospecimen
Antigen in wells was exposed to a combination of sequential treatments. Adsorbed protein was fixed with various concentrations of glutaraldehyde (0.2%, 1%, or 2.5%) along with 4% paraformaldehyde (2.5% in the case of 2.5% glutaraldehyde) made up separately in PBS as well as CB. Then the adsorbed protein was dehydrated up to 70% or 100% alcohol. Separate sets of similarly processed antigen-coated wells were exposed to 60C for 24 hr in a polymerization oven. Dehydration in all cases was carried out by sequentially adding 30%, 50%, 70%, 90%, and 100% ethanol (50 µl/well). After each step the alcohol was flicked off and the next grade of alcohol added (30 min each step and performed at 4C). Separate sets of wells were also processed, in which a 1-hr step of postfixation with 1% OsO4 was included. Finally, a set of aldehyde-fixed, osmium postfixed samples was subjected to antigen retrieval before being assayed by ELISA.
Antigen Retrieval Treatment
Adsorbed antigen that had been fixed with aldehydes and subsequently postfixed with OsO4 was subjected to antigen retrieval by sodium metaperiodate, followed by heating in citrate buffer (
Processing of Cells for TEM
Cells were fixed and processed for embedding in LR White resin as follows:
Antigen Retrieval (Unmasking) Procedure
Sections of samples PBS OsO4 and CBOsO4 were subjected to antigen unmasking.. Sections on grids were incubated in a humid chamber on large drops of fresh saturated sodium metaperiodate for 1 hr at RT. Grids were then floated, sections down, in a beaker containing 0.01 M sodium citrate buffer maintained at 95100C for 15 min.
Immunogold Labeling Method
Sections on grids were first blocked for 30 min, by floating on drops of PBS containing 3% skim milk, 0.01% Tween-20, followed by washing on drops of PBST (five changes, 5 min each). Sections were then incubated overnight (1618 hr) at 4C with rabbit anti-VHb antibody diluted 1:500 in PBS containing 0.3% skim milk and 0.001% Tween-20. Then the grids were washed by floating on drops of PBST. Bound antibodies were visualized by incubating the sections for 1 hr on goat anti-rabbit gold conjugate (10 nm; Sigma) diluted 1:20 in PBS containing 0.3% skim milk and 0.001% Tween-20. Finally, grids were washed on drops of water (five changes, 5 min each) before being stained with 2% aqueous uranyl acetate. All incubations, except for the primary antibody step, were carried out at RT.
Controls
The specificity of the primary antibody reaction was tested by substituting the rabbit anti-VHb antiserum with NRS. The specificity of the primary antibody reaction within tests was also judged by comparing the probe density on the cells with that on the adjacent plastic resin.
Quantitation of Probe Density
The resultant probe density using test and control serum was quantified as follows.
Photography. For each test and normal rabbit immunoglobulin control, the probe density was calculated from random micrographs of cell sections.
Probe Counting.
Prints of negatives were made and overlaid with transparencies. Cell outlines were traced, cut out, and weighed (
Analysis
The total probe density on cells and clear plastic was calculated for the test and control of each sample processed. Densities were expressed as the number of gold particles/µm2. Comparisons between test and control samples was done by two-tailed t test.
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Results |
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Determination of Residual Antigen Binding Activity by ELISA
The effect of various tissue processing steps, independently as well as in combination, for antigen detection by immunogold labeling TEM was studied by an ELISA-based method. Direct protein determination in ELISA wells (
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Single Treatments.
Fixation with 0.2% glutaraldehyde and 4% paraformaldehyde did not result in any loss in the antibody binding activity of the antigen. When the glutaraldehyde concentration was increased to 0.5% and 1% in this mixture, the RA declined to 85% and
60%, respectively. A further increase in the glutaraldehyde concentration to 2.5%, along with 2.5% paraformaldehyde, did not result in any additional alterations in the RA.
When 0.1% tannic acid was included in the primary fixative, the activity dropped to 16% and was undetectable beyond 1% tannic acid. Postfixation with OsO4 after fixation in 0.2% glutaraldehyde and 4% paraformaldehyde resulted in a substantial decrease in RA to approximately 10%.
There was no observable effect of only 30%, 70% and 100% ethanol on antibody binding (Table 1). A drop in the binding activity of samples exposed for 24 hr to 37C (90% RA) and 60C (
75% RA) was noted.
Combination Treatments.
Fixation with 0.2% glutaraldehyde and 4% paraformaldehyde followed by dehydration up to 70% (with or without exposure to 60C) retained the RA at 100%. On the other hand, dehydration up to 100% (with and without heat treatment) in the same fixative resulted in a decline of RA to 85%. A major loss in activity (
10% RA) was observed when the sample was postfixed with osmium. When samples were subjected to antigen retrieval after postfixation, 100% RA was regained.
Fixation in 1% glutaraldehyde and 4% paraformaldehyde and dehydration up to 70% (with or without exposure to 60C) showed the same activity as in the case of single treatment with the same concentration of fixative. Further dehydration up to 100% with heat treatment resulted in a marginal decline of activity when CB was used as vehicle buffer. When the osmium step was included, the RA declined to 5%. Here, recovery in RA was only up to 90% when the sample was subjected to retrieval procedure.
Fixation in 2.5% glutaraldehyde and 2.5% paraformaldehyde and dehydration up to 70% and 100%, with and without exposure to 60C, resulted in similar binding activities (49%).
Nonspecific binding to wells without antigen was negligible in all cases except where post fixation treatment was given. In a set of parallel experiments using control sera, no significant signal was observed in any single or combination of treatments used (data not shown).
Probe Density
The number of gold particles/µm2 of total cell or plastic area are shown in Table 3. The representative areas are shown in Fig 1 Fig 2 Fig 3 Fig 4. On cell sections labeled with anti-VHb antibody, the probe density was always greater than the level of nonspecific binding in adjacent plastic (Fig 1A, Fig 1C, Fig 2A, Fig 2C, Fig 3A, Fig 3C, Fig 4A, and Fig 4C). It was also significantly higher (p<0.001) than the probe density on cell sections similarly treated with normal rabbit serum. (Fig 1B, Fig 1D, Fig 2B, Fig 2D, Fig 3B, Fig 3D, Fig 4B, and Fig 4D).
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Calculation of probe density suggested that immunogold labeling patterns were in agreement with ELISA results regarding retention of antibody binding activity. As observed in ELISA, cells fixed with 1% glutaraldehyde coupled with OSO4 postfixation and dehydration up to 100% ethanol (CBOsO4) demonstrated the lowest probe density of 12.01 particles/ µm2. Probe levels marginally improved to 19.63 particles/µm2 when the primary fixative was replaced with 0.2% glutaraldehyde (PBS OsO4). In cells in which osmium post fixation had been omitted, the probe densities were consistently much higher. As observed in ELISA, lower glutaraldehyde concentrations resulted in higher labeling. Accordingly, the highest probe densities were found in samples that had been fixed with 0.2% glutaraldehyde + 4% paraformaldehyde and in which exposure to ethanol had been limited up to 70% alcohol (PBS70). In this case the probe density was 235.6 particles/µm2. On extending the dehydration up to 100% alcohol (PBS100), the mean probe density dropped significantly (p<0.001) to 160.8 particles/µm2 (30% reduction compared to PBS70). Treatment with 1% glutaraldehyde + 4% paraformaldehyde followed by dehydration up to 70% and 100% alcohol (CB70 and CB100) yielded relatively lower (p<0.001) probe densities of 149.5 and 100.7 particles/µm2, respectively.
Antigen retrieval in osmium-treated samples resulted in a marked regain of probe density to the same levels as obtained with comparable aldehyde fixation and dehydration. In 0.2% glutaraldehyde-fixed cells (PBSOsO4 retrieval), the probe density reverted to 170.9 particles/µm2, and in 1% glutaraldehyde fixation (CBOsO4 retrieval) it increased to 114.6 particles/µm2. In addition, in sections of cells that had been subjected to antigen retrieval, the nonspecific labeling due to NRS increased marginally from 0.24 to 2.08 particles/µm2 for PBS OsO4 retrieval and from 0.44 to 0.59 particles/µm2 for CBOsO4 retrieval. This increase was not significant (p>0.05). In the same sections, the background labeling on plastic also increased slightly. At the dilution used, the gold conjugate by itself did not result in any detectable background labeling on either cells or clear plastic (data not shown).
Cell Structure
Cells fixed with 0.2% or 1% glutaraldehyde in PBS or CB, respectively followed by OSO4 fixation and dehydration up to 100% ethanol, retained clear, well-defined cell structure, with no observable membrane damage. The cytoplasm revealed well-preserved granular and fibrillar structures (Fig 1A1D). When OsO4 postfixation was omitted, the cells showed similar preservation of cytoplasmic structure. However, the membranes were observed to have more folds. Periplasmic spaces also appeared to be more dilated (Fig 2B and Fig 2D). When similarly fixed cells were dehydrated only up to 70% ethanol, significant distortion of morphology was noted. Although the membranes appeared distinct, they were present in a highly wavy form; periplasmic spaces were also seen to be affected. Cytoplasmic fibrillary structures were not so well preserved (Fig 3A3D). Sections that had undergone the retrieval process showed a marginal increase in staining density, with occasional signs of section damage. A slight increase in contamination with particulate debris was also observed in these samples (Fig 4A4D).
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Discussion |
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In this study we have demonstrated the use of ELISA as a convenient method for rapid optimization of antigen detection and quantitation in immunogold labeling electron microscopy. The effects of fixation, by various agents, dehydration, etc. have been extensively studied for their roles in preserving cell structure, as well as their limitation in quantitation of antigen by immunocytochemistry (
We have tested low (0.2%) to high (2.5%) glutaraldehyde concentrations in two commonly used vehicle buffers (PBS and cacodylate) for preliminary screening of residual antigenantibody binding activity by ELISA. It is common practice to use paraformaldehyde along with glutaraldehyde for immunolabeling studies. Therefore, this was also included at 4% concentration in all fixatives, except when the glutaraldehyde concentration was 2.5%. In this case, the concentration of paraformaldehyde was restricted to 2.5% to maintain the total osmotic pressure (1500 mOs). On the basis of the results obtained in ELISA, two concentrations (0.2 and 1%) of glutaraldehyde were selected for final confirmation by immunogold TEM. ELISA results indicated that fixation in a low (0.2%) glutaraldehyde concentration and dehydration up to 70% alcohol, along with exposure to 60C for 24 hr, had practically no effect on the antigen. Presumably, the low levels of fixative stabilize the antigen and prevent its destruction by exposure to 60C. Electron microscopy of the samples processed similarly demonstrated the highest probe density, in agreement with the simulation experiments in ELISA using an artificial immunospecimen. However, TEM revealed distortion of cell morphology in these samples.
The results of ELISA revealed that there was enhanced antigen destruction on combining low glutaraldehyde fixation with exposure to 100% ethanol. However, further heat treatment did not cause any additional loss. On increasing the glutaraldehyde concentration to 1% and 2.5%, antibody binding decreased further. Limiting the dehydration up to 70% ethanol resulted in better retention of antigen than dehydration at a higher gradient. However, when ELISA was carried out with an artificial immunospecimen exposed only to 100% alcohol, the result was no different from exposure to only 70% alcohol (single treatments). This suggests that the additional deleterious effect of 100% alcohol is seen only when combined with prior fixation with 1% (or more) glutaraldehyde. Similarly, as in the case of the ELISA results, quantitative immunogold labeling EM demonstrated that a higher sensitivity of antigen detection was maintained in cells fixed with low glutaraldehyde concentrations. This was also affected by the concentration of alcohol used in dehydration. By increasing the dehydration up to 100% ethanol, it was possible to maintain a high signal and also preserve the cell morphology at a satisfactory level.
Our observations showed that exposure to 100% ethanol resulted in reduction of RA only when this was preceded by a suboptimal fixation with a low concentration (0.2%) of glutaraldehyde. Presumably, the resultant residual aldehyde groups on proteins would fix any non-crosslinked antigen as it undergoes change in conformation upon exposure to a higher gradient of ethanol. In proteins, alcohol-induced conformational changes are known to occur (
When antigen in wells was subjected to only 60C, a 2025% loss of antigen activity was observed, but when similar treatment was given after fixation with 1% glutaraldehyde and dehydration, no additional loss was noted. This may be because the fraction of antigen lost only because of temperature overlaps with the fraction sensitive to 1% or more glutaraldehyde.
Most samples processed for immunolabeling electron microscopy compromise on fixation. The resulting ultrastructure is generally poor compared to tissues processed with conventional methods. This may cause problems in accurate quantitation of label in different cell compartments, in which adequate preservation of cell ultrastructure is important. In this study we have included tannic acid and osmium tetroxide to determine if their effects on immunogold labeling can also be evaluated by ELISA. Because, only a few antigens have been shown to resist exposure to osmium treatment (
The use of 1% OsO4 for postfixation in ELISA caused an approximately 9095% loss in antibody binding. The effect was compounded by use of a higher glutaraldehyde concentration. Unmasking of the fixed antigen resulted in 90100% recovery of activity (when 0.2% glutaraldehyde was used) and 8590% (when 1% glutaraldehyde was used). Electron microscopy showed that the best preservation of structure was obtained when cells were postfixed with osmium, but this resulted in very low levels of labeling. In these cases, subjecting the sections to retrieval resulted in elevation of the total labeling densities on cells to the same level as that of cells fixed with the same concentration of aldehydes and subjected to dehydration up to 100% in alcohol (p>0.05), thus demonstrating a total recovery of antigen lost during osmium postfixation, similar to our ELISA findings. This was accompanied by an increase in background labeling on the plastic which, however, did not pose a problem for quantification of the cellular antigen because the ratio of number of particles/µm2 on cells and the number of particles/µm2 on plastic was >10. Nonspecific labeling by NRS on cells increased very slightly and was also significantly lower than test samples (p<0.001).
Although the techniques of fixation, the nature of the fixative solutions, and the embedding procedure can interfere with the cytochemical demonstration of tissue components, not all components are altered, and the effect varies according to the nature of the target binding site. In general, the use of low concentrations of fixatives has resulted in good cytochemical labeling (
Our results have shown that the trend of residual antibody binding activity of antigen, adsorbed in ELISA wells (artificial immunospecimen), after a simulated processing for electron microscopy, is similar to that obtained by immunogold labeling electron microscopy. Therefore, it can provide information regarding antigen loss through simulation of various EM tissue processing steps. This method offers significant advantages over the existing method using nitrocellulose membranes. Apart from the fact that ELISA-based methods are inherently more sensitive for quantitation (instead of merely detection) of antigen, it also offers the following benefits: (a) It is possible to study the effect of organic solvents such as alcohol. (b) The effect of osmium postfixation and retrieval techniques can be easily assayed in a quantitative manner. (c) Our method utilizes antigens in their native conformation as opposed to antigens that have been denatured by boiling with sodium dodecyl sulfate before electrophoretic transfer to the membrane (
Our assay system utilizes a cell lysate that can be prepared more easily instead of the purified antigen that has been suggested for dot-blot assays. Moreover, when pure antigen is used the effect of crosslinking fixative is not truly mimicked because the antigen is not surrounded by other proteins present in the original sample (
Although we have worked with a soluble intracellular antigen, in principle the ELISA technique can also be applied to other proteins, including membrane-bound antigens (
In carrying out immunogold quantitation of antigens via electron microscopy, crucial decisions about the exact processing method must be made. This is especially so when the sample is available in limited quantity. Screening by ELISA would therefore assist in making a logical decision about the antigen losses due to processing steps and thereby aid in the selection of an optimal protocol for immunogold labeling electron microscopy.
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Acknowledgments |
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We wish to thank Drs A. Mondal, R. Kishore, G.C. Varshney, and A. Mukhopadhyay for critical reading and correction of the manuscript. The skillful technical assistance of Mr Anil Theophilus is gratefully acknowledged. This is IMTech Communication no. 019/2000.
Received for publication July 26, 2000; accepted November 1, 2000.
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