BRIEF REPORT |
Correspondence to: Harald Jockusch, Developmental Biology and Molecular Pathology, W7, University of Bielefeld, D-33501 Bielefeld, Germany. E-mail: h.jockusch@uni-bielefeld.de
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Summary |
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Green fluorescent protein (GFP) and its variants, such as enhanced GFP (EGFP), have been introduced into mammalian cells by transgenes, e.g., to distinguish donor from host cells after transplantation. Free GFP is extremely soluble and leaks out from liquid-covered cryostat sections so that fixation of whole organs before sectioning has been mandatory. This precludes the analysis of serial sections with respect to fixation-sensitive enzyme activities and antigens. We describe here a vapor fixation for sections from unfixed cryostat blocks of tissue that allows unrestricted enzyme and immunohistochemistry on adjacent sections, as demonstrated for cross-striated muscle and other tissues from EGFP transgenic "green mice" and for a transplantation experiment.
(J Histochem Cytochem 51:401404, 2003)
Key Words: green fluorescent protein, transgenes, vapor fixation
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Introduction |
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IN EXPERIMENTAL EMBRYOLOGY and transplantation research, it is important to distinguish cell and tissue contributions from different sources, e.g., the two partners used to produce embryo aggregation chimeras or donor from host in the case of cell or organ grafts. Stable genetic differences are preferable to vital dye or radioactive labeling because they are not diluted by cell division, and histological markers (like nuclear, cytoplasmic, or membrane differences) are more informative than biochemical markers (such as alloenzymes) that require homogenization of the tissue to be analyzed. In recent years, transgenic labels have been used extensively, in particular bacterial ß-galactosidase localized in the cytoplasm (LacZ transgene) or in the nucleus (nLacZ) and green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its artificial variants such as enhanced GFP (EGFP). GFP has the advantage of being a "vital dye" whose presence can be monitored in living organisms and whole unfixed organs, by appropriate UV illumination (
In this study we used "green mice" provided by Professor Masaru Okabe (Osaka University) through Professor Melitta Schachner (ZMNH Hamburg). These mice are transgenic for an enhanced green fluorescent protein (EGFP, hitherto designated "GFP") construct in which the GFP cDNA is downstream to the chicken ß-actin promoter. Non-GFP controls were NMRI mice (HarlanWinkelmann; Borchen, Germany). Whole organs were either shock-frozen in liquefied propane (-190C) or fixed in an excess of 3.7% formaldehyde and calcium- and magnesium-free PBS (CMF-PBS) for 24 hr at 4C with gentle rocking ("bath fixation," the method recommended by Okabe; http://133.1.15.131/tg/greenmouse.cfm), followed by 24 hr in CMF-PBS. Organs from GFP and non-GFP ("0") mice were tied together with sewing thread before fixation to yield "artificial chimeric blocks." In one case, a specimen from a transplantation experiment was used (
Cryostat sections (8-µm thick) from unfixed frozen tissues were fixed for 20 min under a drop of 3.7% formaldehyde in CMF-PBS and then processed conventionally ("drop fixation"). For vapor fixation, cryostat sections on SuperFrostPlus adhesive microscope slides (MenzelGläser; Braunschweig, Germany) were dried for about 5 min and then exposed, in a tightly closed plastic dish, for 212 hr at -20C to the vapor of filter paper soaked with 37% formaldehyde (commercial stock solution; Sigma, F-1268, Dreieich, Germany). Sections were usually counterstained with Hoechst 33342 (1 µg/ml; Sigma, B 2261) and embedded in Elvanol (10 g Mowiol 4-88 in 40 ml CMF-PBS and 20 ml glycerol). Laminin and myosin heavy chain (MyHC) immunostaining was performed with a polyclonal anti-laminin (Sigma; L-9393) using an appropriate horseradish peroxidase-labeled second antibody which was developed with diaminobenzidine (DAB) and a monoclonal anti-MyHC MF20 (
Methods of fixation of GFP-expressing tissues were compared with respect to the following parameters: overall morphology, retention of GFP in GFP-positive tissue regions, "overspill of label" from GFP-positive to neighboring unlabeled tissue, and the possibility of parallel analysis of the same tissue block. To this end, we have produced, from several organs that express high levels of GFP "chimeric blocks" that consisted of pairwise arrangements GFP-labeled and unlabeled tissues in close contact: skeletal muscle, cardiac muscle, stomach, and pancreas (Fig 1).
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The bath fixation served as the positive control. Retention of GFP in all tissues was optimal and there was no overspill. Cryostat sections from prefixed skeletal muscle, however, were more difficult to handle than unfixed cryostat sections, and muscle fibers showed a tendency to shrink (Fig 1A vs 1C). The possibility to perform enzyme and immunohistochemistry on adjacent sections was drastically reduced because of the previous fixation, as exemplified by SDH and anti-laminin staining (Fig 1D vs 1E).
Embedding in paraffin greatly improves the quality of sections and yields high resolution (
In tissues with smaller cells, in which the cytoplasmic area exposed to liquid fixative is not as large as in skeletal muscle fibers, such as smooth muscle of stomach and secretory cells in pancreas, liquid fixation of cryostat sections gave surprisingly poor results, whereas vapor fixation showed high retention, low background, and little if any marginal overspill (Fig 1K1N).
As a possible application, the distinction of host from donor muscle cells after transplantation of the myogenic cell line C2C12 under the kidney capsule of a GFP nude mouse is shown (Fig 1F1H). Because mature and regenerating host myofibers were found close to the transplantation site, their distinction from donor-derived myotubes was crucial. Whereas the former were unambiguously GFP-positive, the latter were GFP-negative.
The following observations have been made with regard to the differential expression of GFP in our stock of Okabe's green mice. All types of muscleskeletal, cardiac, and smoothshowed very high levels of GFP expression. In mixed muscle, the oxidative small-diameter IIA fibers had somewhat higher GFP levels than large-diameter glycolytic IIB fibers (Fig 1C and Fig 1E, arrows). Embryonic and neonatal skeletal muscle had lower GFP levels than adult muscle (
Skeletal muscle is an exceptionally favorable material to be studied in serial sections because it is easy to identify and follow individual fibers over several hundred micrometers. It is now possible to combine this traditional technique with the modern tool of GFP labeling. In contrast to whole-organ fixation, vapor fixation of single cryostat sections allows an unlimited application of standard immunocytochemical methods to adjacent sections in which cells of interest have been recognized by the GFP label. In comparison to bath fixation with subsequent paraffin embedding, the resolution at the subcellular level may be lower, but this is limited anyway by the high solubility of GFP in the cytoplasm. In the most important biomedical model organism, the mouse, our method can be applied to cell or organ transplantation (
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Acknowledgments |
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We thank Professor Masaru Okabe (Osaka University) for providing his "green mice," and Dr. P. Heimann and D. Kanig for help with image and text processing.
Supported by the Deutsche Forschungsgemeinschaft, Graduate Program 231 "Strukturbildungsprozesse," and SFB 549 "Processing of and Signalling by Extracellular Macromolecules," and by Fonds der Chemischen Industry (FCI).
Received for publication July 18, 2002; accepted October 24, 2002.
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