TECHNICAL NOTE |
Correspondence to: Roeland W. Dirks, Dept. of Molecular Cell Biology, Lab. of Cytochemistry and Cytometry, Leiden University, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.
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Summary |
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Using RNA in situ hybridization to reveal cytoplasmic localization patterns of mRNAs in cultured cells, we noted unexpected staining of a cytoplasmic component in telophase cells. Control experiments revealed that the anti-digoxin-specific antibody was responsible for this staining. Because the staining was observed only at a position where both daughter cells are still connected, we identified the stained component as the midbody. This was confirmed by double staining of cells with anti-digoxin and anti--tubulin antibodies. We concluded that anti-digoxin-specific antibody shows crossreactivity with a component present in the midbody. (J Histochem Cytochem 46:779782, 1998)
Key Words: digoxin, midbody, crossreactivity, RNA in situ hybridization
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Introduction |
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In situ hybridization (ISH) techniques make use of hapten- or fluorochrome-labeled probes to detect nucleic acid sequences in biological preparations. Although convenient in multicolor hybridization experiments, direct visualization of fluorochromized probes often provides lower detection sensitivity compared to hapten-labeled ones (
For many years we have successfully used digoxigenin-labeled probes in conjunction with anti-digoxigenin and anti-digoxin antibodies in our studies to specifically detect mRNA molecules at the light and electron microscopic levels (reviewed in -tubulin. These results suggest a nonspecific binding of the anti-digoxin antibody to one of the midbody components.
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Materials and Methods |
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Cell Lines
X1 cells (HeLa cells containing a luciferase gene construct), human foreskin fibroblasts, and a rat fibroblast cell line (rat 9G) were grown on glass microscopic object slides at 37C in Dulbecco's minimal essential medium without phenol red (Gibco; Gaithersburg, MD), supplemented with 10% fetal calf serum, antibiotics, and glutamine (also from Gibco).
In Situ Hybridization
The in situ hybridization procedure was performed as described previously (
Immunocytochemical Detection
Antibodies used in this study were MAbs anti-digoxigenin (Boehringer Mannheim; Mannheim, Germany), FITC-conjugated anti-digoxin (clone no. DI-22; Sigma), anti--tubulin (Amersham; Poole, UK), and polyclonal antibody sheep anti-digoxigeninFITC (Boehringer Mannheim). Cells were incubated with 1:500 dilutions of these antibodies in TBS (150 mM NaCl, 100 mM Tris-HCl, pH 7.2) containing 0.5% (w/v) blocking reagent (Boehringer Mannheim) for 30 min. Next, cells were washed three times in TBS for 5 min each and incubated for 30 min with rabbit anti-mouseFITC (Sigma) diluted 1:500. After three washes in TBS, slides were embedded in Vectashield (Vector Labs; Burlingame, CA) containing 4',6'-diamidino-2-phenyl indole (DAPI). For double staining, cells were first incubated with anti-tubulin followed by an incubation with goat anti-mouseCy3 (Jackson Immunoresearch; West Grove, PA). Then cells were incubated with normal mouse serum followed by an incubation with mouse anti-digoxinFITC.
Cells were examined with a DM epifluorescence microscope (Leica) equipped with appropriate filter sets for red, green, and blue single excitation and double or triple excitation filters for multicolor exposures. Digital images were captured with a cooled CCD camera (Photometrics) and image analysis was performed on a Macintosh computer using SCIL image (Multihouse, The Netherlands).
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Results and Discussion |
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ISH experiments designed to visualize mRNA distribution patterns in different cultured cell lines (see Materials and Methods) using digoxigenin-labeled probes and MAb anti-digoxin revealed an unexpected "specific" staining of a cytoplasmic component exclusively present in telophase cells. Figure 1 shows an example of such staining in X1 cells hybridized with a probe for ß-actin mRNA. In addition to the small fluorescent spots indicating ß-actin mRNA, strong staining is present at the sites where both cells are still attached to each other (Figure 1). Control experiments omitting the digoxigenin-labeled probe revealed that this strong staining was derived from the anti-digoxin-specific antibody. Furthermore, this staining pattern was not observed when the ß-actin probe was labeled with biotin and detected with a streptavidin conjugate. Although anti-digoxin shows 100% crossreactivity with digoxigenin, the anti-digoxigenin-specific antibodies tested in this study did not reveal any staining of cytoplasmic components in telophase cells. When cells were hybridized first with a Cy3-labeled oligonucleotide probe specific for poly (A)+ tails or 28S rRNA sequences and then incubated with FITC-labeled anti-digoxin MAb, it appeared that the cellular component stained by this antibody did not contain RNA sequences (Figure 2a and Figure 2b).
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Because staining with anti-digoxin MAb was observed only in telophase cells at a position where both daughter cells were still connected to each other, it is most likely that the stained component is the midbody. The midbody is identified at the EM level as amorphous electron-dense matrix material and consists of microtubules and at least 35 proteins (-tubulin.
-Tubulin has been identified as one of the major components of the midbody (
-tubulin. Spindle figures in metaphase and anaphase cells also stained positively for
-tubulin but did not stain with anti-digoxin MAb, precluding crossreactivity with
-tubulin (result not shown). Apparently, this antibody shows crossreactivity with one of the other midbody's proteins.
Apart from being used in hybridization studies to detect digoxigenin-labeled probes (e.g.,
In conclusion, anti-digoxin MAb shows crossreactivity with one of the midbody's components. Because this crossreactivity is confined to a defined body present in telophase cells only, it will in general not trouble interpretation of ISH results. Furthermore, this crossreactivity is not observed with anti-digoxigenin-specific antibodies. Digoxigenin will therefore remain one of the most useful haptens to be employed in ISH studies.
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Acknowledgments |
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Supported in part by Boehringer Mannheim.
Received for publication September 15, 1997; accepted January 22, 1998.
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