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RAPID COMMUNICATION |
Simultaneous Detection of EGFP and Cell Surface Markers by Fluorescence Microscopy in Lymphoid Tissues
Kim L. Kussera and Troy D. Randallaa Trudeau Institute, Saranac Lake, New York
Correspondence to: Troy D. Randall, Trudeau Institute, P.O. Box 59, 100 Algonquin Ave., Saranac Lake, NY 12983. E-mail: trandall@trudeauinstitute.org
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Summary |
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 Top Summary IntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Enhanced GFP (EGFP) is a powerful tool for the visualization of tagged proteins and transfected cells and is easily detected by fluorescence microscopy or flow cytometry in living cells. However, soluble EGFP molecules can be lost if cell integrity is disrupted by freezing, sectioning, or permeablization. Furthermore, the fluorescence of EGFP is dependent on its conformation. Therefore, fixation protocols that immobilize EGFP may also destroy its usefulness as a fluorescent reporter. Here we determined which methods of preparing murine lymphoid tissues immobilized soluble EGFP protein and retained its fluorescence while simultaneously maintaining the antigenicity of various immunologically important molecules and best preserving the overall morphology of the tissues. We found that EGFP could not be visualized in frozen sections of spleen that had not been fixed before freezing. However, robust EGFP fluorescence could be observed in frozen sections of tissues fixed under various conditions. Fixation was important to immobilize EGFP rather than to maintain conformation, because only minimal EGFP could be detected by immunofluorescence in unfixed frozen sections. Although it had little effect on EGFP fluorescence, the inclusion of sucrose during fixation better preserved the morphology of fixed tissues. These methods also preserved the antigenicity of a wide variety of molecules used to identify cell types in lymphoid tissues. (J Histochem Cytochem 51:5–14, 2003)
Key Words: EGFP, frozen sections, immunofluorescence, mouse, spleen, perfusion, cell surface markers
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Introduction |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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GREEN FLUORESCENT PROTEIN (GFP) is a naturally fluorescent gene product of the jellyfish Aequorea victoria. Native GFP protein is excited by UV light (395 nm) and is not particularly bright (
Because the fluorescence of EGFP is independent of other proteins, co-factors, or substrates (
The advantage of each of these systems is that the fluorescence of EGFP unambiguously tags recombinant proteins, marks transfected cells, or reports gene expression without steps that interfere with the integrity of the cells or that might introduce background or nonspecific fluorescence. Although the visualization of EGFP in living cells is sufficient for many procedures, other applications require the simultaneous detection of EGFP and other molecules. Unfortunately, soluble EGFP molecules are inefficiently retained in tissue sections or in cells that have lost membrane integrity (
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Materials and Methods |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Mice
EGFP transgenic mice were generated as described (
Cell Transfers
Spleens were removed from EGFP transgenic mice and mechanically disrupted by passage through wire mesh. Single-cell suspensions were depleted of erythrocytes by incubation in 150 mM NH4Cl, 1 mM KHCO3, 0.1 mM EDTA, pH 7.3, for 5 min. The remaining leukocytes were washed and resuspended in 2% FCS in PBS. Recipient mice were injected IV with 1–5 x 107 splenocytes, depending on the experiment.
Fixation Procedures
For fixation by immersion, spleens were removed from recipient mice and cut in half. One half was saved for analysis by flow cytometry and the other half was fixed by immersion in either 4% paraformaldehyde or 4% formalin in PBS, with or without 7% picric acid and with or without 10% sucrose, as indicated in Table 1. The time of fixation varied from 30 min to 24 hr. For fixation by perfusion, mice were injected IP with 100 U heparin (Elkins–Sinn; Cherry Hill, NJ) and sacrificed by CO2 inhalation 20 min later. The thoracic cavity was opened and a 25-gauge needle was inserted into the right ventricle and connected directly to a 20-ml syringe for manual perfusion, or by 0.8-mm internal diameter Tygon tubing (BioRad; Hercules, CA) to a 20-ml syringe at a height of 45 cm for gravity perfusion. The inferior vena cava was cut to allow outflow of fixative. Mice were first perfused with 20 ml of 2 U/ml heparin in PBS. This was followed by a second perfusion with 20 ml of either 4% paraformaldehyde or 4% formalin in PBS, with or without 7% or 14% picric acid, as indicated in Table 2. Mice were perfused a final time with 20 ml of 10% sucrose in PBS.
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Decalcification of NALT
Mice that had been fixed by perfusion were decapitated and the skin, lower jaw, and incisors of the upper jaw were removed. The remaining tissue was placed in a rotating 50 ml tube with 7% EDTA and 10% sucrose in PBS. Decalcification took place over several days and the EDTA solution was changed daily.
Embedding and Sectioning
Fresh or fixed spleens were frozen directly in Tissue-Tec OCT (Fisher; Pittsburgh, PA) above liquid nitrogen. Fixed and decalcified heads were equilibrated in OCT over time by placing the heads in a rotating 50 ml tube with graded concentrations of OCT in PBS (25%, 50%, 75%, and 100%). Heads were embedded in OCT and frozen over liquid nitrogen. Frozen tissue blocks were brought to –20C and 7-µm sections were cut and placed on poly-l-lysine-coated slides. Slides were dried at room temperature (RT) overnight, then either probed with antibodies or stored at –20C.
Immunofluorescence
Slides were brought to RT and fixed in acetone at 4C for 10 min. Slides were then placed in PBS for 5 min to remove the OCT. All slides were blocked with 5% BSA in PBS for 30 min. Endogenous avidin and biotin were blocked with the Avidin-Biotin Blocking kit from Vector (Burlingame, CA). Slides were washed three times for 5 min each in PBS before being probed with biotinylated antibodies to various antigens for 30 min in a humidified chamber. Biotinylated anti-B220 (RA3-6B2), anti-CD3 (145-2c11), anti-Thy-1.2 (53-2-1), anti-CD11c (HL3), anti-CD21/CD35 (8C12), and anti-IgD (217-170) were purchased from PharMingen (San Diego, CA). Biotinylated peanut agglutinin (PNA) was obtained from Vector. Alexa 594-conjugated rabbit anti-GFP was purchased from Molecular Probes (Eugene, OR). After incubation with biotinylated antibodies, slides were washed three times for 5 min in PBS. They were then incubated with streptavidin–Alexa 594 (Molecular Probes) for 30 min, washed three times for 5 min each in PBS, and mounted with Polymount (Polysciences; Warrington, PA). In some experiments, sections were mounted with SlowFade Light antifade kit containing DAPI (Molecular Probes).
Microscopy and Digital Imaging
Slides were viewed with a Zeiss Axioplan 2 microscope using a 480/30 bandpass filterset to view EGFP, a 560/40 bandpass filter set to view Alexa 594, and a 330/80 filter set to view DAPI. Images were recorded with a Zeiss AxioCam digital camera (Zeiss; Thornwood, NY) using the Zeiss proprietary software, Axiovision 3.0.6.0. Images were manipulated in Adobe Photoshop 5.5 and saved as final figures in TIFF format at 600 DPI.
Flow Cytometry
In experiments in which spleens were not fixed by perfusion, the engraftment of EGFP transgenic cells into recipient spleens was monitored by flow cytometry. Half spleens were disrupted by passage through wire mesh and single-cell suspensions were obtained. Erythrocytes were lysed by 5-min incubation in 150 mM NH4Cl, 1 mM KHCO3, 0.1 mM EDTA, pH 7.3, and the remaining leukocytes were washed and resuspended in 2% FCS in PBS with 1 µg/ml propidium iodide. Cells were analyzed using a FACSCalibur (Becton–Dickinson; Mountain View, CA) flow cytometer. Dead cells were excluded from analysis by the fluorescence of propidium iodide in the FL3 channel and EGFP-expressing cells were identified in the FL1 channel.
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Results |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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To determine if the fluorescence of EGFP-expressing cells was retained in frozen sections, we transferred splenocytes from EGFP transgenic mice to nontransgenic recipients. EGFP transgenic mice express EGFP constitutively in all tissues under the control of the chicken actin promoter and CMV enhancer. All leukocytes in these mice express uniformly high levels of EGFP and are easily distinguished from nontransgenic cells by FACS (not shown). Recipients were sacrificed 2 days after transfer and spleens removed for analysis. Half of each spleen was snap-frozen in OCT medium over liquid nitrogen, while the remaining tissue was mechanically disrupted and made into a single cell suspension for analysis by flow cytometry. Frozen tissues were sectioned (7 µm), air-dried, and fixed in acetone. Sections were then probed with biotinylated anti-B220 antibodies followed by streptavidin–Alexa 594 and analyzed by fluorescence microscopy. As shown in Fig 1A, whereas B220-expressing cells could be easily detected (red), no EGFP-expressing cells could be observed in snap-frozen sections, even though a substantial population of EGFP-expressing cells could be observed by flow cytometry (Fig 1A, inset). Because it was previously suggested that slow freezing of tissues better preserved EGFP fluorescence in frozen sections (
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We next tested whether a cryopreservative, such as sucrose, maintained EGFP fluorescence in frozen sections. Therefore, we adoptively transferred EGFP-expressing cells into nontransgenic recipients for 2 days, immersed recipient spleens in 10% sucrose at 4C overnight, and embedded tissues in OCT by snap-freezing above liquid nitrogen. Sections from frozen tissues were probed with anti-B220 and analyzed by fluorescence microscopy. Again, no EGFP expression was detectable (Fig 1C), even though EGFP-expressing cells were clearly present in the tissue as visualized by flow cytometry (Fig 1C, inset).
We reasoned that the failure to detect EGFP expression was due either to the instability of the EGFP fluorophore in sections of frozen tissues or to the loss of the soluble EGFP from the sectioned cells. Therefore, to stabilize the fluorophore and to immobilize the soluble EGFP within the cells, we tried various methods of fixation before sectioning and analysis. As before, we transferred splenocytes from EGFP transgenic mice into nontransgenic recipients. Mice were sacrificed 2 days after transfer and spleens were removed and cut in half. Half of each spleen was mechanically disrupted and analyzed by flow cytometry to demonstrate the presence of EGFP-expressing cells, and the remainder of the tissues was fixed by immersion for 30 min, 2 hr or 24 hr at 4C in either 4% paraformaldehyde or 4% formalin in PBS, with or without 10% sucrose as a cryopreservative and with or without 7% picric acid as an additional fixative. Fixed tissues were then embedded in OCT and snap-frozen over liquid nitrogen. Frozen tissues were sectioned (7 µm), fixed in acetone, and probed with anti-B220 before analysis by fluorescence microscopy. As shown in Fig 1D, EGFP-expressing cells could be clearly observed in sections of tissues that had been fixed for 24 hr in 4% paraformaldehyde in PBS. The addition of 10% sucrose to this fixation protocol had little effect on EGFP fluorescence (Fig 1E and Fig 1F). Similarly, EGFP-expressing cells could be observed in sections of tissues fixed for 24 hr in 4% formalin in PBS (Fig 1G). Again, the addition of 10% sucrose did not dramatically affect EGFP fluorescence (Fig 1H and Fig 1I). Finally, EGFP-expressing cells could be observed in sections of tissues fixed for 24 hr with the combination of 4% formalin and 7% picric acid (Fig 1J), regardless of whether or not sucrose was added (Fig 1K and Fig 1L). Therefore, fixation by at least three different methods preserved the fluorescence of EGFP in frozen sections.
Although fixation preserved the fluorescence of EGFP, the morphology of the fixed tissues (Fig 1D and Fig 1L) was different from that of tissues frozen directly (Fig 1A–1C). Whereas cells in sections of unfixed spleens were plump and tightly packed (Fig 1A–1C), cells in sections of fixed spleens were somewhat desiccated in appearance and there were gaps between the cells (Fig 1D, Fig 1G, and Fig 1J). The addition of sucrose during the fixation process partially alleviated this problem (Fig 1E, Fig 1H, and Fig 1K). In particular, the addition of sucrose helped maintain cell shape (shown at higher magnification in Fig 1F, Fig 1I, and Fig 1L), even though there were still gaps between the cells.
To determine whether soluble EGFP was simply lost from sections of unfixed tissues or whether the protein was retained in a conformation that precluded fluorescence, we probed frozen sections of unfixed tissues with antibodies to GFP. As shown in Fig 1M and Fig 1N, EGFP was barely detectable with anti-GFP antibodies in sections of tissues that had been snap-frozen (Fig 1M) or slow-frozen (Fig 1N), even though EGFP-expressing cells were clearly present by FACS analysis (insets). However, anti-GFP antibodies clearly detected EGFP in frozen sections of tissues previously fixed in 4% formalin/7% picric acid/10% sucrose (Fig 1O). In fact, all cells expressing EGFP were also detected by the anti-GFP antibody (Fig 1O, inset). Therefore, we conclude that the primary reason for loss of EGFP fluorescence in unfixed frozen sections is that soluble EGFP molecules are inefficiently retained in sections of tissues that are not fixed before freezing.
The results from the above experiments are summarized in more detail in Table 1. As discussed above, the presence and fluorescence of EGFP were maintained by all fixatives (Table 1), although we felt that the combination of 4% formalin and 7% picric acid in PBS consistently provided the brightest EGFP signal. Furthermore, the inclusion of sucrose consistently improved the morphology of the tissue, even though it had little effect on EGFP fluorescence (Table 1). Finally, the timing of fixation had little effect on morphology or EGFP fluorescence, although extended periods of fixation tended to compromise the binding of some antibodies, particularly anti-IgD (not shown).
To clarify whether the fixation conditions that best preserved both EGFP fluorescence and cell morphology also maintained the ability of various monoclonal antibodies to recognize their respective antigens on frozen sections, we transferred splenocytes from EGFP transgenic mice to a nontransgenic recipient and after 2 days we prepared half of the spleen for analysis by flow cytometry and fixed the remainder in 4% formalin, 7% picric acid, 10% sucrose in PBS for 2 hr before freezing, sectioning, and analysis by immunofluorescence. As shown in Fig 2, this fixation procedure maintained robust EGFP fluorescence and preserved the ability of multiple antibodies to bind to fixed sections. For example, anti-CD3 could be used to detect discrete T-cell areas in the spleen (Fig 2A). As shown at higher magnification in Fig 2B, many of the transferred EGFP-expressing splenocytes also expressed CD3 and were found in the T-cell area. Similarly, rounded B-cell follicles were observed with anti-B220 (Fig 2C and Fig 2D) and with anti-IgD (Fig 2E and Fig 2F). Furthermore, many of the transferred EGFP-expressing splenocytes also expressed B220 or IgD and migrated to the B-cell follicles (Fig 2D and Fig 2F). Reticular networks of follicular dendritic cells (FDCs) could also be found within the B-cell follicles as visualized with anti-CD21 (Fig 2G and Fig 2H). Although transferred EGFP-expressing cells were found adjacent to FDCs, none of the FDCs themselves expressed EGFP (Fig 2H), consistent with their stromal rather than hematopoietic origin (
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We next tested whether fixation by perfusion, rather than immersion, resulted in better EGFP fluorescence, antibody binding, and morphology. Therefore, we transferred EGFP-transgenic cells to nontransgenic recipients and sacrificed the recipients 2 days later. After sacrifice, mice were perfused through the right ventricle with 20 ml of heparin in PBS, followed by 20 ml of either 4% paraformaldehyde or formalin in PBS, with or without picric acid at various concentrations, as indicated in Table 2. Mice were finally perfused with 20 ml of 10% sucrose in PBS. The spleens were then excised, frozen in OCT over liquid nitrogen, and sectioned. As shown in Table 2, the fluorescence of EGFP was maintained in all of the perfusion conditions tested, although it was somewhat brighter in mice perfused with 4% formalin and 7% picric acid, similar to what we observed with the immersion fixation method (Table 1). Although the rate of perfusion did not affect the fluorescence of EGFP (Fig 3, insets), it markedly affected the morphology of the tissue (Fig 3). Rapid manual perfusion tended to displace B-lymphocytes from the follicles and to scatter them throughout the splenic matrix (Fig 3A). This was observed to a lesser extent with slow manual perfusion (Fig 3B) and was minimized by gravity perfusion (Fig 3C). However, even the gentle perfusion pressure induced by gravity resulted in cell displacement compared to immersion-fixation (Fig 2C) or snap-freezing (Fig 1A–1C). Again, the inclusion of sucrose maintained cell shape (Fig 3).
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We also tested the ability of various antibodies to bind frozen sections of perfusion-fixed spleens. As shown in Table 2, all of the tested antibodies were able to detect their respective antigens on sections from spleens fixed under each of the tested conditions. This was somewhat surprising, considering that we had difficulty detecting IgD in sections of spleens fixed by immersion with similar reagents (not shown). However, other experiments suggested that the epitope recognized by the anti-IgD antibody is destroyed by longer fixation times. Therefore, the difference between the immersion-fixation and perfusion-fixation experiments is likely due to the length of time that the tissue is fixed rather than to the reagents used. Because tissues are in contact with fixative for less than an hour during perfusion, the epitope recognized by the anti-IgD antibody is maintained.
We next determined whether EGFP fluorescence was maintained through the prolonged process of fixation and decalcification required to section nasal associated lymphoid tissue (NALT) (
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Although the visualization of EGFP in living cells usually poses few problems (
Other reports have demonstrated that the fluorescence of EGFP is maintained in frozen sections of fixed tissues (
The requirement for a fixative suggested that soluble EGFP was simply leaking out of the sectioned cells. This hypothesis was supported by data showing that EGFP could not be detected by anti-GFP antibodies in unfixed frozen sections (Fig 1M–1O). However, our attempts to immobilize EGFP after sectioning by fixing sections with acetone, formalin, or paraformaldehyde were uniformly unsuccessful (not shown), suggesting that the loss of soluble EGFP occurs rapidly after thawing. The loss of EGFP is particularly acute in our experimental system, in which cells from mice transgenic for a soluble form of EGFP are adoptively transferred to nontransgenic recipients. In other systems, in which EGFP is fused to protein domains that are anchored to the cytoskeleton, have transmembrane regions, or are otherwise immobilized in the cell, the loss of EGFP may not be as severe.
Although the loss of EGFP signal in our experimental system is clearly related to loss of EGFP protein rather than to a change in the conformation of EGFP, some procedures do appear detrimental to EGFP conformation and fluorescence. For example, we have found that EGFP fluorescence decreases with extended periods of fixation (not shown). Furthermore, other investigators have demonstrated that the fluorescence of EGFP is lost on paraffin embedding of paraformaldehyde-fixed tissues (
Although multiple methods of fixation were able to preserve the fluorescence of EGFP, the fixation process often resulted in cells with a desiccated appearance and gaps between cells. This problem was partially overcome by the addition of 10% sucrose to the fixative. With the addition of sucrose, cells retained their rounded shapes, even though some gaps remained between the cells. In addition, although the perfusion-fixation method was best suited for uniform fixation of tissues that were not easily immersed, such as the NALT, perfusion often resulted in disrupted splenic architecture, particularly at high rates of perfusion (Fig 3). This is due to the direct connection of the spleen to the blood. The architecture of other lymphoid organs, such as the NALT, lymph nodes, and Peyer's patches, were less affected by perfusion pressure because they are continuous with lymphatics rather than with blood vessels.
Fixation of lymphoid organs under conditions that best preserve the fluorescence of EGFP also maintains the ability of multiple antibodies to be used for immunofluorescence (Fig 2). These antibodies are used to define the architecture of lymphoid organs, which consists of separated B- and T-cell areas and their associated dendritic cells. Therefore, the placement of EGFP-expressing cells in lymphoid organs can be easily determined using these methods.
In summary, we describe a method of preparing murine lymphoid tissues that optimally immobilizes EGFP and preserves its fluorescence, while simultaneously allowing the concurrent visualization of multiple cell surface markers and maintaining the cell architecture of the spleen and other lymphoid organs. Although we have demonstrated the utility of this method to detect adoptively transferred EGFP-expressing cells, similar methods could also be used to detect EGFP produced under the control of gene-specific or tissue-specific promoters.
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Acknowledgments |
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Supported by the Trudeau Institute and by NIH grant AI43589 (to T.D.R.).
We would like to thank Dr Frances Lund for critically reviewing this manuscript.
Received for publication July 23, 2002; accepted August 23, 2002.
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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B. O. Lee, J. Moyron-Quiroz, J. Rangel-Moreno, K. L. Kusser, L. Hartson, F. Sprague, F. E. Lund, and T. D. Randall CD40, but Not CD154, Expression on B Cells Is Necessary for Optimal Primary B Cell Responses J. Immunol., December 1, 2003; 171(11): 5707 - 5717. [Abstract] [Full Text] [PDF]
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