ARTICLE |
Correspondence to: Toshihiro Takizawa, Dept. of Physiology and Cell Biology, Ohio State University, 304 Hamilton Hall, Columbus, OH 43210. E-mail: takizawa.2@osu.edu
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Summary |
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Here we show that ultrathin cryosections of placental tissue can be used as a substrate in immunofluorescence experiments. A high degree of spatial resolution can be achieved in these preparations because there is essentially no out-of-focus fluorescence. Therefore, immunofluorescence microscopy using ultrathin cryosections provides a very useful method for determining the precise subcellular localization of antigens in tissues. In addition, ultrathin cryosections of placenta also serve as a substrate for correlative immunofluorescence and immunoelectron microscopy using FluoroNanogold as the detection system. In correlative microscopy, the exact same structures in the same ultrathin section were observed by both fluorescence and electron microscopy. Using a particle counting procedure and electron microscopy, we compared the labeling obtained with colloidal gold and FluoroNanogold and found a higher number of particles with silver-enhanced FluoroNanogold than with colloidal gold. (J Histochem Cytochem 51:707714, 2003)
Key Words: placenta, immunocytochemistry, correlative microscopy, ultrathin cryosections, caveolin, early endosome antigen 1, FluoroNanogold
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Introduction |
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IMMUNOCYTOCHEMISTRY provides a unique set of methodologies for determining the distribution of antigens in cells and tissues. Methods exist for the detection of antigens at both the light and the electron microscopic level. Detection of antigenantibody binding requires a reporter that can be visualized in light and/or electron microscopes. The most commonly used reporter systems are fluorochromes, enzymes, and particles (e.g., colloidal gold). Detection of antigenantibody binding can be direct (the primary antibody contains a reporter) or indirect (the primary antibody is unlabeled and the secondary immunoprobe contains the reporter).
In many studies, determination of which cells in a population (e.g., a tissue section) are positive for a particular antigen will suffice. However, in other cases more precise determination of the distribution of an antigen in a particular cell may be required. In simple tissue culture systems, relatively precise localization can be accomplished with immunofluorescence microscopy (IFM) methods, and very precise localization can be achieved with immunoelectron microscopy (IEM). In organized tissues, precise localization of antigens can be achieved by IEM. However, precise localization of antigens in tissues by IF methods may be problematic. Conventionally, tissues are sectioned at 515-µm thickness with a cryostat before IFM localization. Such sections can be examined with a conventional fluorescence microscope, in which case the fluorescence from the entire volume of the section will be observed. The out-of-focus fluorescence that may be in the section leads to image degradation and loss of resolution. Alternatively, the sections can be examined with a confocal microscope, in which case optical sectioning permits examination of a restricted volume of the cryostat section. This limits most out-of-focus fluorescence and yields enhanced resolution compared to conventional fluorescence microscopy (e.g.,
We have described methods for correlative fluorescence and electron microscopy of isolated neutrophils (
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Materials and Methods |
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Reagents
Alexa Fluor 488- and 594-labeled goat anti-mouse and goat anti-chicken antibodies, and goat anti-rabbit antibodies, 4',6-diamidino-2-phenylindole (DAPI), as well as the ProLong antifade kit were obtained from Molecular Probes (Eugene, OR). Alexa Fluor 594-labeled streptavidin FluoroNanogold (FNG) was from Nanoprobes (Yaphank, NY). Biotin-labeled goat anti-chicken F(ab)'2 and goat anti-chicken 4-nm colloidal gold antibodies were from Jackson ImmunoResearch (West Grove, PA). Fetal bovine serum was obtained from Invitrogen (Carlsbad, CA). Gelatin was obtained from Sigma Chemical (St Louis, MO). All immunological reagents were handled in accordance with the manufacturers' recommendations and were used within the expiration date for each product. A murine monoclonal antibody (MAb) to early endosome antigen 1 (EEA 1) was obtained from BD Transduction Laboratories (San Diego, CA). An anti-peptide antibody specific for caveolin-1 (CAV-1
) was generated in chickens. The immunogen was amino acids 314 from the N-terminal region of the human CAV-1
molecule with the amino acid sequence GGKYVDSEGHLY. Characterization of this antibody has been reported (
Processing Ultrathin Cryosections for Immunocytochemistry
Human full-term placentae were used as the model tissue and were obtained from the Ohio State University tissue procurement service according to a protocol approved by the Ohio State University Human Subjects Institutional Review Board. Tissue samples from cesarean deliveries were processed for fixation as rapidly as possible (no more than 15 min after delivery). Three placentae were used in this study.
Tissue was cut into small pieces and fixed in 4% paraformaldehyde (freshly prepared) in 0.1 M sodium cacodylate buffer, pH 7.4, containing 5% sucrose for 2 hr at room temperature (RT). The samples were washed and embedded in 10% gelatin in the same buffer. The solidified gelatin was cut into smaller pieces and then cryoprotected by infiltration with 2.3 M sucrose in 0.1 M sodium cacodylate (pH 7.4) overnight at 4C. After sucrose infiltration, the samples were mounted on specimen pins designed to fit a Reichert cryoultramicrotome, frozen in liquid nitrogen, and stored in liquid nitrogen until they were sectioned.
Ultrathin cryosections were cut with a Reichert Ultracut E equipped with an FC 4D cryounit. Sections (100-nm thickness or less) were cut on a Cryo P diamond knife (Diatome-US; Fort Washington, PA) and were collected on droplets of 0.75% gelatin2.0 M sucrose (
Immunofluorescence Microscopy
Ultrathin cryosections were mounted on glass coverslips when only IFM microscopy was done. Coverslips containing sections were immersed in a solution containing 1% non-fat dry milk and 5% fetal bovine serum in PBS (MFBSPBS) for 15 min at 37C to remove the sucrose and gelatin, then washed three times in PBS and incubated in MFBSPBS to block nonspecific protein binding sites; 0.05% sodium azide was also present. Ultrathin cryosections were then incubated with chicken anti-CAV-1 (diluted 1:500 in MFBSPBS) and anti-EEA 1 (diluted to 10 µg/ml) for 30 min at 37C. The cryosections were rinsed in PBS four times over 15 min, immersed in MFBSPBS, and then incubated with Alexa 488-labeled goat anti-chicken IgG and Alexa 594-labeled goat anti-mouse IgG (both diluted 1:200 in MFBSPBS) for 30 min at 37C. After immunolabeling, the cryosections were washed five times in PBS. The sections were then incubated with DAPI (1 µg/ml) in PBS for 10 min at RT in order to visualize nuclei. The sections were then washed five times in PBS and mounted on glass microscope slides in ProLong antifade medium. Control sections received the same treatment except for omission of the primary antibody or substitution of pre-immune chicken antibody for the primary antibody.
Correlative Microscopy
Ultrathin cryosections were collected on nickel Maxtaform "finder" grids (Graticules; Tonbridge, Kent, UK) to facilitate location of specific structures when going from the optical to the electron microscope. Sections on grids were incubated in the same manner as were the 12-mm coverslips containing ultrathin cryosections. After washing in PBS and MFBSPBS (the same as for ultrathin cryosections on coverslips), the grids were incubated with biotin-labeled goat anti-chicken (13 µg/ml in MFBSPBS) for 30 min at 37C. The grids were then washed in PBS for 12 min with four changes and then immersed in MFBSPBS. The grids were next incubated with streptavidin-labeled FNG conjugated with Alexa 594 (diluted 1:50 in MFBSPBS) for 30 min at RT. The grids were then washed in PBS for 15 minutes with five changes.
After the labeling procedure, the grids were mounted on a glass microscope slide in PBS containing 1% N-propyl gallate and 50% glycerol, pH 8.0, to retard photobleaching (
Immunoelectron Microscopy with Colloidal Gold
Ultrathin cryosections were incubated for the localization of CAV-1 with colloidal gold as the reporter system. Sections on EM grids were incubated with the primary antibody and then washed in the same manner as previously described for IFM. The sections were then incubated with goat anti-chicken 4-nm colloidal gold diluted 1:10 in MFBSPBS for 30 min at 37C. The sections were then washed with five changes of PBS over 15 min and then fixed in 2% glutaraldehyde for 30 min. The sections were then washed in distilled water for 6 min with four changes. They were then subjected to the same positive contrast procedure that was used for the correlative microscopy.
Microscopy
Fluorescence and differential interference contrast (DIC) images were collected with a Nikon Optiphot microscope equipped with a Photometrics Cool Snap fx CCD camera (Roper Scientific; Tucson, AZ). Images were captured with Metamorph image analysis software system (Universal Imaging; Dowingtown, PA). All fluorescence images were collected in the monochrome mode and then converted to the appropriate pseudo-color where needed. Electron microscopy was carried out with a Philips CM-12 transmission electron microscope operated at 60 kV. Figures shown in this report were compiled with Photoshop 7 software (Adobe Systems; Mountain View, CA).
Quantitative Analysis of Immunogold Labeling
Electron micrographs of CAV-1 localization that was detected with 4-nm colloidal gold or with silver-enhanced FNG were analyzed by counting particles associated with caveola-like structures. A total of 100 randomly selected caveola were scored for each type of detection system.
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Results |
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Immunofluorescence in Ultrathin Cryosections
Ultrathin cryosections of human placenta (70100-nm thickness) were tested as a substrate for IFM microscopy using a chicken anti-CAV-1 and an MAb directed against EEA 1 as the primary antibodies. Detection of anti-CAV-1
and anti-EEA 1 binding was achieved with goat anti-chicken Alexa 488 and goat anti-mouse Alexa 594 as the secondary antibodies (Fig 1). In ultrathin cryosections, CAV-1
was present in small punctate structures in the capillary endothelial cells and pericytes but not in the syncytiotrophoblast (STB) layer of terminal villi of the placenta. These fluorescent structures are suggestive of individual caveolae or small groups of caveolae. The distribution of EEA 1 was decidedly different. It was located primarily in vesicle-like structures in the apical portion of the STB. Relatively few EEA 1-positive structures were observed in endothelial cells or pericytes. These structures were not observed in control incubations when pre-immune antibody replaced the specific antibody for CAV-1 or when the primary antibody was omitted (CAV-1 and EEA 1) (data not shown). These results demonstrate that ultrathin cryosections of placenta serve as an excellent substrate for high-resolution IFM labeling.
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Correlative Immunofluorescence and Immunoelectron Microscopy in Ultrathin Cryosections
Ultrathin cryosections were used as a substrate for correlative IFM and IEM. Ultrathin cryosections collected on EM grids were immunolabeled with the chicken anti-CAV-1. The detection of anti-CAV-1
binding to the section was achieved with biotin-labeled goat anti-chicken antibody followed by Alexa 594 FNGstreptavidin binding (Fig 2A). The IFM distribution of CAV-1
in placenta, as determined with FNG as the reporter system, appears identical to that obtained with conventional fluorochrome-tagged secondary antibodies (compare Fig 1 and Fig 2A). The morphology of the ultrathin cryosection on the EM grid was determined by DIC microscopy (Fig 2B). After collection of the fluorescence and DIC images, the FNG was subjected to the silver enhancement procedure to render the FNG visible at the EM level. Electron microscopy showed that caveolae were now heavily decorated with silver-enhanced gold particles (Fig 2D). These gold-labeled caveolae correspond precisely to the fluorescence signal from FNG once the fluorescence images are at the same magnification as the EM images (Fig 2C).
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Comparison of the Labeling Achieved with Fluorochrome-, Colloidal Gold-, and FNG-labeled Secondary Immunoprobes
The detection of chicken anti-CAV-1 binding to ultrathin cryosections of terminal villi of human placenta was demonstrated with (a) fluorochrome-, (b) colloidal gold-, and (c) FNG-labeled secondary immunoprobes (Fig 3). As far as we can tell, all caveolae were detected with fluorochrome-labeled secondary antibodies. Colloidal gold-labeled secondary antibodies also labeled caveolae. However, in this case not all of the caveola-like structures were labeled. Moreover, the signal was relatively weak, with about two colloidal gold particles per caveolae-like structure (Table 1). When silver-enhanced FNG was the detection system, all caveola-like structures appeared to be labeled. In addition, these structures were heavily decorated with about 27 particles per caveola (Table 1).
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Discussion |
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Immunofluorescence microscopy has been a valuable tool in experimental and applied biomedicine. It provides a high degree of sensitivity for the in situ detection of antigens while being a relatively simple technique. The adequacy of spatial resolution that can be achieved with IFM depends on the questions being addressed. In situations where the question of interest relates to which cells in a population (e.g., tissue section) are positive, spatial resolution is less important than is sensitivity of detection. In other cases, where the subcellular location of an antigen within the positive cell is of interest, then spatial resolution is as important as sensitivity of detection. In the first situation, positive cells can be distinguished from negative cells in conventional cryostat sections of tissue (e.g., 510-µm thickness) by IFM. However, these types of cryostat sections are not adequate for resolution of subcellular detail by IFM (e.g., to caveolae in the placenta (
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Immunoelectron microscopy provides a very high degree of spatial resolution but is generally less sensitive than is IFM. However, IEM does provide a "reference space," i.e., all structures are visible, not just the immunolabeled components. In IFM, on the other hand, the "reference space" is absent; in IFM, only the labeled structures are observable, and the unlabeled structures are not observed and do not contribute to the analysis (
We referred above to enhanced sensitivity of detection of antigen localization in light microscopy compared to electron microscopy (i.e., fluorescence vs colloidal gold). We tested this proposal using ultrathin cryosections of placenta for the localization of CAV-1 (see Fig 3). When fluorochrome-labeled secondary antibodies were used, individual caveola were detected; the fluorescence was associated with the entire caveola. On the other hand, when 4-nm colloidal gold-labeled secondary antibodies were used, the caveola were sparsely labeled with about two colloidal gold particles per caveola. Moreover, the caveolae were not uniformly labeled using colloidal gold. Some caveola-like structures were unlabeled (not shown). Detection of CAV-1
binding to ultrathin cryosections was also achieved with FNG as the detection system. In IFM, qualitatively similar results were obtained when fluorochrome-labeled secondary antibodies were used or when FNG was used as the detection system (compare Fig 1A and Fig 2A). After silver enhancement of FNG, the caveolae were heavily labeled. Moreover, all caveola-like structures were labeled (see Fig 2D and Fig 3C). There were significantly more particles associated with caveolae when FNG was the detection system compared to colloidal gold. Moreover, with FNG the same structures detected by IFM were also detected by IEM. In summary, we show that ultrathin cryosections of tissue serve as an excellent substrate for IFM and for correlative IFM and IEM.
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Footnotes |
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1 Portions of this work were presented at a workshop on the Use of Gold and Gold/Fluorescence for Improved Histology at the Sixth Joint Meeting of the Japan Society of Histochemistry and Cytochemistry and the Histochemical Society in Seattle, WA, July 2002.
Received for publication January 27, 2003; accepted January 29, 2003.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Burry RW (1995) Pre-embedding immunocytochemistry with silver enhanced small gold particles. In Hayat MA, ed. Immunogold-silver Staining. Principles, Methods, and Applications. Boca Raton, FL, CRC Press, 217-230
Griffith JM, Posthuma G (2002) A reliable and convenient method to store ultrathin thawed cryosections before immunolabeling. J Histochem Cytochem 50:57-62
Griffiths G (2001) Bringing electron microscopy back into focus for cell biology. Trends Cell Biol 11:153-154[Medline]
Griffiths G, Parton RG, Lucocq J, van Deurs B, Brown D, Slot JW, Geuze HJ (1993) The immunofluorescence era of membrane traffic. Trends Cell Biol 3:214-219[Medline]
Inoué S (1995) Foundations of confocal scanned imaging in light microscopy. In Pawley JB, ed. Handbook of Biological Confocal Microscopy. New York, Plenum Press, 1-17
Ishiko A, Shimizu H, Masunaga T, Kurihara Y, Nishikawa T (1998) Detection of antigens by immunofluorescence on ultrathin cryosections of skin. J Histochem Cytochem 46:1455-1460
Liou W, Geuze HJ, Slot JW (1996) Improving structural integrity of cryosections for immunogold labeling. Histochem Cell Biol 106:41-58[Medline]
Lyden TW, Anderson CL, Robinson JM (2002) The endothelium but not the syncytiotrophoblast of human placenta expresses caveolae. Placenta 23:640-652[Medline]
Majlof L, Forsgren PO (1993) Confocal microscopy: important considerations for accurate imaging. Methods Cell Biol 38:79-95[Medline]
Pawley J (1995) Fundamental limits in confocal microscopy. In Pawley JB, ed. Handbook of Biological Confocal Microscopy. New York, Plenum Press, 19-37
Pombo A, Hollinshead M, Cook PR (1999) Bridging the resolution gap: imaging the same transcription factories in cryosections by light and electron microscopy. J Histochem Cytochem 47:471-480
Powell RD, Halsey CM, Hainfeld JF (1998) Combined fluorescent and gold immunoprobes: reagents and methods for correlative light and electron microscopy. Microsc Res Tech 42:2-12[Medline]
Robinson JM, Takizawa T, Pombo A, Cook PR (2001) Correlative fluorescence and electron microscopy on ultrathin cryosections: bridging the resolution gap. J Histochem Cytochem 49:803-808
Robinson JM, Takizawa T, Vandré DD (2000) Applications of gold cluster compounds in immunocytochemistry and correlative microscopy: comparison with colloidal gold. J Microsc 199:163-179[Medline]
Robinson JM, Vandré DD (2001) Antigen retrieval in cells and tissues: enhancement with sodium dodecyl sulfate. Histochem Cell Biol 116:119-130[Medline]
Rothberg KG, Heuser JE, Donzell WC, Ying Y-S, Glenney JR, Anderson RGW (1992) Caveolin, a protein component of caveolae membrane coats. Cell 68:673-682[Medline]
Takata K, Kasahara T, Kasahara M, Ezaki K, Hirano H (1991) Localization of Na+-dependent active type and erythrocyte/HepG2-type glucose transporters in rat kidney: immunofluorescence and immunogold study. J Histochem Cytochem 39:287-298[Abstract]
Takizawa T, Anderson CL, Robinson JM (2003) A new method to enhance contrast of ultrathin cryosections for immunoelectron microscopy. J Histochem Cytochem 51:31-39
Takizawa T, Robinson JM (1994) Composition of the transfer medium is crucial for high-resolution immunocytochemistry of cryosectioned human neutrophils. J Histochem Cytochem 42:1157-1159
Takizawa T, Robinson JM (2000) Analysis of antiphotobleaching reagents for use with FluoroNanogold in correlative microscopy. J Histochem Cytochem 49:433-436
Takizawa T, Robinson JM (2003) Correlative microscopy of ultrathin cryosections is a powerful tool for placental research. Placenta 24:559-567
Takizawa T, Suzuki K, Robinson JM (1998) Correlative microscopy using FluoroNanogold on ultrathin cryosections: proof of principle. J Histochem Cytochem 46:1097-1102
Tokuyasu KT (1980) Immunochemistry on ultrathin frozen sections. Histochem J 12:381-403[Medline]
Tokuyasu KT (1983) Present state of immunocryoultramicrotomy. J Histochem Cytochem 31:164-167[Medline]
Tokuyasu KT, Maher PA, Singer SJ (1972) Distribution of vimentin and desmin in developing chick myotubes in vivo. I. Immunofluorescence study. J Cell Biol 98:1961-1972
Zucker RM, Price O (2001) Evaluation of confocal microscopy system performance. Cytometry 44:273-294[Medline]