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1 Wolfson Centre for Inherited Neuromuscular Disease, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, SY10 7AG, UK
2 Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK
3 UMRS 787, Institute of Myology, INSERM-Universite Paris 6, 75013 Paris, France
4 Institute for Science and Technology in Medicine, Keele University, Keele, ST5 5BG, UK
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Abstract |
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Introduction |
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Aberrant RNA transcripts arising from genetic mutations in myotonic dystrophies also result in the production of nuclear foci and offer an opportunity to study formation of foci when specific mRNAs are processed through normal pathways. Myotonic dystrophy type 1 (DM1) is caused by the expansion of a trinucleotide (CTG) repeat in exon 15 in the 3'-untranslated region (UTR) of the myotonic dystrophy (DM) protein kinase (DMPK) gene on chromosome 19q13.3 (Brook et al. 1992; Fu et al. 1992; Mahadevan et al. 1992). A second form of DM (DM2) is due to the expansion of a tetranucleotide (CCTG) repeat in intron 1 of the zinc finger protein 9 (ZNF9) gene on chromosome 3q21.3 (Liquori et al. 2001). Transgenic mice models with expanded CUG repeats in the 3'-UTR of the unrelated muscle-specific actin or the human DMPK transcripts were found to develop features of DM1 (Mankodi et al. 2000; Seznec et al. 2001), suggesting that the major clinical features of DM1 are directly due to the repeat expansion. The expanded repeats in DM1 and DM2 remain in the nuclei as discrete ribonuclear inclusions or foci (Taneja et al. 1995; Hamshere et al. 1997; Liquori et al. 2001) and contain double-stranded hairpin loop structures (Jasinska et al. 2003; Kino et al. 2004). Mutant DMPK pre-mRNA in DM1 is spliced and polyadenylated normally (Davis et al. 1997). Mutant ZNF9 pre-mRNA in DM2 is also spliced normally, and the mRNA is exported from the nucleus (Margolis et al. 2006). Ribonuclear inclusions in DM1 and DM2 therefore arise because of different levels of processing. DM1 foci are composed of mRNA, whereas DM2 foci consist of tetranucleotide repeat sequences lacking most, or all, of the flanking intronic RNA (Margolis et al. 2006).
Muscleblind, or MBNL, proteins bind specifically to expanded, but not normal size, CUG repeats in a manner proportional to repeat size (Miller et al. 2000). Human MBNL isoforms (MBNL1, MBNL2 and MBNL3) co-localize with the expanded CUG/CCUG ribonuclear inclusions in DM cells (Miller et al. 2000; Fardaei et al. 2001, 2002; Mankodi et al. 2001, 2003; Jiang et al. 2004). They are RNA-binding proteins that regulate alternative splicing (reviewed by Pascual et al. 2006) and are themselves subject to alternative splicing (Fardaei et al. 2002). At least some of the pathological features of DM may be due to misregulated alternative splicing of RNA (reviewed by Osborne & Thornton 2006). An MBNL1 knockout mouse shows DM features, such as myotonia, abnormal myofibers, cataracts and aberrant splicing of the chloride channel, cardiac troponin T and fast skeletal troponin T (Kanadia et al. 2003), suggesting that sequestration of MBNL1 has a role in DM pathogenesis.
In the present study, we show for the first time a significant association between speckle structures and nuclear foci of DMPK mRNA with expanded CUG repeats, whereas expanded CCUG repeats derived from intronic RNA show no such association. We suggest that nuclear foci form when the RNAs containing these repeats are recognized as "defective" and are blocked at some stage in their nuclear export or turnover.
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Results |
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Figure 1 shows that the mAb, MB1a, recognizes a band of the expected size (42 kDa) on Western blots of total HeLa cell protein extracts. The mAb did not cross-react with MBNL2 or MBNL3 isoforms (Fardaei et al. 2002), as judged both by Western blots of recombinant proteins and by immunolocalization of transfected COS-7 cells (data not shown). The mAb also stained nuclear foci, co-localizing with CUG repeats in DMPK mRNA identified by in situ hybridization in cultured DM1 myoblasts (Fig. 2A). Rarely, nuclear spots occurred, which were either labelled with antibody or probe but not both. We assume that this was due to incomplete penetration of antibody or probe. In situ hybridization is greatly facilitated by the large numbers of CUG repeats (800+) binding the labelled (CAG)10 probe in these cells, derived originally from congenital DM1 fetuses. Detection of endogenous DMPK mRNA without repeats (e.g. from the non-mutant allele) is not possible with the method used.
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Because of the possible role of MBNL proteins in nuclear RNA splicing, we examined the relationship between nuclear DM1 foci and splicing-related nuclear structures. We confirmed earlier reports of the lack of any significant co-localization of nuclear foci with nuclear "gems" (using mAbs against SMN, gemins 6 and 7 as markers (Young et al. 2000; Sharma et al. 2005)) or with nuclear Cajal bodies (using mAb against coilin p80 as a marker (Bohmann et al. 1995) (results not shown). Figure 2 shows that there is also no co-localization of nuclear DM1 foci with structures containing YT521-B, a protein involved in alternative splicing (Nayler et al. 2000; Fig. 2B) or with "paraspeckles" (Fox et al. 2002; Fig. 2C). In order to do double-label experiments between MBNL1 and other mouse mAbs, we prepared a rabbit antiserum against MBNL1 and Fig. 2D shows that this antibody identifies the same nuclear foci as the MB1a mAb.
Nuclear foci in DM1 cells are located at the periphery of splicing speckles labelled by Y12, 7.13, SC35 or 9G8 antibodies
Figure 3A shows a significant association in DM1 myoblasts between nuclear foci stained for MBNL1 and nuclear speckles stained by the mAb, Y12. The Y12 epitope is now known to include a symmetrical dimethylarginine present in the Sm core proteins of snRNPs (Brahms et al. 2000) and in the Cajal body protein, coilin (Boisvert et al. 2002), but similar results were obtained when an antibody against a specific splicing factor, 9G8 protein (Cavaloc et al. 1994), was used to label nuclear splicing speckles. Figure 4A,B show the association between 9G8- and (CAG)10-labelled CUG repeats in DM1 myoblast and fibroblast nuclei, respectively. The use of 9G8 antibody in this study is further validated by its co-localization with SC35 protein (Fig. 4D), which has become a widely used marker for nuclear speckles (Fu & Maniatis 1990).
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In DM2 cells, the nuclear foci do not associate with either speckles or exosomes
In DM2 fibroblasts, there was no association of nuclear foci with Y12 structures using either in situ hybridization with (CAGG)10 (Fig. 3E) or MBNL1 pAb (Fig. 3F). Figure 4C confirms the lack of association of DM2 foci with (CAGG)10 repeats using mAb 9G8 as the speckle marker. Because exosomes are involved in the turnover of intronic RNA after splicing (Bousquet-Antonelli et al. 2000), we looked for association between ribonuclear foci and the exosome in DM2 cells, but none was found (Fig. 4E).
Quantitative studies confirm a significant association of expanded repeats with splicing speckles in DM1 but not in DM2
Table 1 shows results of counting the ribonuclear foci that appeared to be associated with the Y12 speckles. The percentage of foci associated with Y12 structures was high in DM1 myoblasts and fibroblasts (ca. 90%) and was not altered by treatment with actinomycin D (P = 0.5). The area of the nucleus occupied by Y12 staining decreased from 35.9% in untreated myoblasts to 20.2% in actinomycin D-treated myoblasts (P < 0.001). The area of Y12 staining was also around 20% in DM1 and DM2 fibroblasts after actinomycin D treatment. The percentage of foci associated with Y12 structures in DM2 fibroblasts was also as low as 20% and therefore likely to be due to chance. In DM1 cells, however, the association between nuclear foci and Y12 structures was much greater (90%) than that would be expected by chance (P < 0.001; Table 1). This analysis shows that the pictures in Fig. 3 are representative, but we also needed a more objective method of assigning foci as "associated" or "not associated" with speckles. Quantitative analysis of confocal images of the cells using IMAGEJ software was used to measure the distances between Y12 speckles and nuclear foci (Table 2). Threshold levels were adjusted so that the area of nuclear Y12 staining was decreased to between 1% and 2% of the total nuclear area in order to identify the centres of speckles. The average distances from each ribonuclear focus to its nearest speckle center were similar in untreated and actinomycin D-treated DM1 myoblasts, respectively, and 0.7 µm in DM1 fibroblasts (Table 2). This is consistent with our average "center-to-edge" measurement for speckles of 0.7 µm (range: 0.4–1.7 µm; data not shown). This speckle-to-focus distance increased significantly in DM2 fibroblasts to 1.8 µm (P < 0.001), consistent with a lack of association in DM2. The average distance between each speckle center and its nearest neighboring speckle centre was 2.6 µm. These measurements objectively confirm the differences between DM1 and DM2 nuclei that can be seen in Figs 3 and 4.
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Figure 5 shows a schematic and hypothetical model that explains our observations in a simplified way. Both DM1 and DM2 are caused by repeat expansions in the DMPK and ZNF9 genes, respectively. In DM1, the expansion is in an exon and it inhibits the normal export of DMPK mRNA, whereas in DM2, the expansion is in an intron and the ZNF9 mRNA is exported normally, but turnover of the spliced out intron is inhibited (Fig. 5A). Figure 5B illustrates the accumulation of mutant DMPK mRNA, not only near the single site of transcription close to a splicing speckle but also at several more distant speckles (Taneja et al. 1995). Our hypothesis is that DMPK mRNA would normally progress on the export pathway beyond the speckle periphery (see Discussion) but is prevented from doing so by the expanded repeats and accumulates at the point where the normal pathway is blocked. In DM2, processed ZNF9 mRNA having lost its expanded intronic repeats can be exported normally, though whether this particular mRNA passes through the speckle is not known (hence, the question marks in Fig. 5B). The intronic repeats still accumulate as foci in DM2, presumably because the normal turnover pathway is inhibited. This hypothesis prompted us to look at exosomes which may be involved at some stage in the turnover of at least some introns, but this is evidently not the site of CCUG repeat accumulation (Fig. 4E).
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Y14 protein is a component of the exon-junction complex which binds to RNA transcripts during splicing. Y14 remains associated with spliced mRNA during export to the cytoplasm by the mRNA transport protein NXF1 (TAP). Y14 is mainly associated with nuclear speckles but sometimes extends beyond them into the perispeckle region (Schmidt et al. 2006). Does Y14 remain with mutant DMPK mRNA in nuclear foci (Y14 concentrated in nuclear foci) or does it dissociate from this non-exportable mRNA after splicing (Y14 absent from foci)? Figure 6A shows that Y14 is associated with DM1 nuclear foci but not preferentially concentrated at the foci so the answer is not clear-cut. The possibility that failure to bind exporter proteins contributes to the failure to export the mutant mRNA has not yet been excluded. The splicing factor hnRNP-H, which interacts with mutant DMPK mRNA when both CUG repeats and a distal splice site are present, also promotes nuclear retention (Kim et al. 2005) and can interact directly with MBNL1 in pull-down experiments (Paul et al. 2006). Figure 6B shows that hnRNP-H, a known component of nuclear speckles (see Discussion), is more widespread than Y14 in the nucleoplasm, extending well beyond the speckle regions to include the areas occupied by nuclear foci. Figure 6B shows that nuclear foci are usually close to, and often overlap, areas of strong endogenous hnRNP-H staining. In agreement with previous observations (Kim et al. 2005; Paul et al. 2006), most of the hnRNP-H is not associated with RNA foci, but this does not exclude a role for hnRNP-H in nuclear retention of DMPK mRNA.
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Discussion |
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Inhibition of RNA synthesis for 4 h with actinomycin D reduced the area of the nucleus occupied by speckles, as previously reported (Lamond & Spector 2003), but did not alter their association with nuclear DM1 foci. About 90% of the foci were adjacent to Y12 speckles in both treated and untreated DM1 cells (Table 1), showing that the association with the speckle periphery does not require continued RNA synthesis. There was little loss of in situ hybridization intensity over this period, indicating that mutant DMPK mRNA is lost only slowly from the foci when further synthesis is blocked.
The RNA transcripts in DM1 foci are processed DMPK mRNA, without introns, polyadenylated and containing the expanded CUG repeats in the 3'-UTR (Hamshere et al. 1997). In DM2, on the other hand, mutant ZNF9 pre-mRNA is also spliced and polyadenylated, but the mRNA is exported to the cytoplasm where normal levels of ZNF9 protein expression occur (Margolis et al. 2006). The DM2 ribonuclear foci contain only the CCUG repeat sequence derived from intron 1 but with no detectable flanking intronic RNA (Margolis et al. 2006). This is illustrated schematically in Fig. 5A. Our data suggest that expanded repeat mRNA in DM1 accumulates at specific locations in the nucleus at some stage after splicing and polyadenylation but before transport to the nuclear pore for export into the cytoplasm as mRNP particles (reviewed in Erkmann & Kutay 2004). The region in electron micrographs immediately adjacent to IGCs or speckles is thought to consist of perichromatin fibrils, which are the sites of transcription and splicing (Fakan 1994). The specific location of DMPK mRNA foci at the boundaries of speckles in DM1 is not simply due to aggregation of repeats immediately after transcription from the gene in the adjacent chromatin; this is because Taneja et al. (1995) showed that only one of the multiple foci in each DM1 nucleus is associated with DMPK DNA sequences at transcription sites, though all foci contain DMPK mRNA sequences. This suggests that some of the DMPK mRNA has relocated from its transcription site to form foci at several other sites adjacent to speckles (Fig. 5B). There is evidence that endogenous mRNA sequences are capable of rapid diffusion within the nucleoplasm (Molenaar et al. 2004; Politz et al. 2006). It is not yet clear whether relocation of mutant DMPK mRNA from the site of transcription to the more distant speckles occurs at the pre-mRNA stage or after processing into mRNA, since both origin and destination are in mRNA-processing regions of the nucleus. Custodio et al. (1999), however, showed that ß-globin pre-mRNA was only partially spliced at the site of transcription, splicing being completed elsewhere in the nucleus, so it is possible that multiple nuclear foci in DM1 cells correspond to sites where DMPK mRNA splicing is completed.
A further question for consideration is whether the location of DM1 foci adjacent to speckles represents a blocked location in the normal mRNA export pathway or, alternatively, a blocked stage in the turnover pathway for "defective" mRNAs. There is evidence in yeast for a close interrelationship between the formation of an exportable mRNP, 3' polyadenylation, mRNA degradation and the release of mRNA from the site of transcription (Stutz & Izaurralde 2003). Thus, an essential role has been proposed for exosomes in a surveillance mechanism that decides whether a newly formed mRNA is exportable or defective (Vasudevan & Peltz 2003; Erkmann & Kutay 2004). Interestingly, "defective" mRNAs often accumulate in nuclear foci, similar to DM1 foci, and these persist when transcription is inhibited (Erkmann & Kutay 2004). On the other hand, there is also evidence for accumulation of "normal" mRNAs in foci adjacent to speckles when normal export pathways are inhibited. Thus, micro-injected fushi tarazu pre-mRNA was exported from HeLa nuclei, but the spliced mRNA was degraded, suggesting a close link between RNA transcript processing and tagging for export as mRNA (Tokunaga et al. 2006).When transcription was inhibited, the micro-injected fushi tarazu pre-mRNA accumulated as spliced mRNA at discrete foci adjacent to speckles. The location of DM1 foci is clearly not determined by MBNL1 because this is present in both DM1 and DM2 foci. The possibility that some intrinsic property of CUG repeats, not shared by CCUG repeats, enables them to bind directly to a partner present only in the perispeckle region cannot be ruled out. It seems more likely that the location is determined by the mRNA itself, since other mRNAs, without repeats, can form similar foci under some circumstances. Tiscornia & Mahadevan (2000) identified four splicing factors, including known speckle components hnRNP-C, U2AF and PSF (Saitoh et al. 2004), that associate with the DMPK mRNA 3'-UTR close to a splice site downstream of the CUG repeats. This binding appeared to enable splicing of the expanded CUG repeats in DM1 and consequent release of the block on DMPK mRNA export from the nucleus and production of a novel DMPK protein isoform (Tiscornia & Mahadevan 2000). It is interesting that DMPK mRNA containing expanded CUG repeats accumulates in foci close to a source of splicing factors that can release it from nuclear retention, since this would be consistent with a blocked export pathway hypothesis. A role for hnRNP-H in the nuclear retention of mutant DMPK transcripts in DM1 has been proposed, since it interacts with DMPK mRNA when both CUG repeats and a distal splice site are present (Kim et al. 2005) and also interacts with MBNL1 (Paul et al. 2006). It is interesting to note that an early description of hnRNP-H showed it to be present in nuclear speckles (Matunis et al. 1994), and a recent proteomic study of purified IGCs confirmed this (Saitoh et al. 2004), although it is also present elsewhere in the nucleoplasm (Fig. 6B). Furthermore, Y12 anti-speckle antibodies have also been used to purify hnRNP-H by co-immunoprecipitation (Chou et al. 1999). The precise relationship between hnRNP-H in speckles and hnRNP-H associated with DMPK mRNA in DM1 remains unclear. However, it is at least a plausible hypothesis that the accumulation of mutant DMPK mRNAs adjacent to speckles is linked to release from the speckles of splicing factors that interact with the mutant mRNA.
Several recent studies have suggested that many, but not all, mRNAs pass through the speckle for export (Smith et al. 1999; Melcak et al. 2000; Hattinger et al. 2002; Shopland et al. 2002), including micro-injected fushi tarazu pre-mRNA after processing (Tokunaga et al. 2006). Furthermore, the complex between Y14 and NXF1 (TAP), which marks nuclear mRNAs as "export-competent," was also found mainly within speckles, supporting some role for speckles in mRNA export (Schmidt et al. 2006). This would explain why we found nuclear foci adjacent to both speckles (Figs 3 and 4) and Y14 protein staining (Fig. 6A). It seems unlikely that expanded CUG repeats interfere directly with binding of the Y14 complex, but this remains to be established. The mechanism of mRNA transport between the site of transcription and processing (perichromatin fibrils) and the site of export into the cytoplasm (nuclear pores) may not be the same for all mRNAs (Politz et al. 2006).
The possibility that nuclear focus formation in DM is driven by interaction and aggregation of the expanded repeat sequences cannot be ruled out, but there is little direct evidence for this. Similar foci are produced when processing of different mRNAs without repeats is inhibited or when mRNAs are defective in other ways (Erkmann & Kutay 2004; Tokunaga et al. 2006). Although it is now clear that nuclear CUG-repeat foci in DM1 are non-randomly distributed in the nucleus, this is less clear for CCUG-repeat foci in DM2. The latter appear randomly distributed with respect to speckles, but association with some as-yet-unidentified structure cannot be ruled out. The formation of DM2 foci by expanded CCUG repeats alone could be simple aggregation, but it is also consistent with accumulation of long RNA hairpins at sites where intron degradation is blocked or retarded. Further study of the location and mechanism of formation of nuclear foci in DM1 and DM2 may throw more light on RNA processing pathways in the nucleus. Although altered RNA splicing appears to be a direct cause of some clinical features of DM (Osborne & Thornton 2006), the location of DM1 foci close to splicing speckles may not be relevant to this, since the clinical features of DM1 and DM2 are quite similar.
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Experimental procedures |
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Human MBNL1 cDNA was amplified from pEGFP/MBNL1 (Fardaei et al. 2001) using primers 5'-GCGGATCCCGTCACACCAATTCGGGA-3' and 5'-GCGTCGACGTCAGATGTTCGGCAGATATTATGG-3' with BamHI and SalI sites (underlined) for cloning into pET21b. After transformation of Escherichia coli BL21(DE3) and induction with IPTG, bacterial pellets were washed by sonication in TNE buffer and recombinant protein was extracted by sonication in 6 M urea in PBS. MBNL1 was purified by His-tag column chromatography in 6 M urea.
The recombinant protein was used as the immunogen for production of mAbs using the hybridoma method (Nguyen & Morris 2002). Polyclonal rabbit antiserum to MBNL1 was prepared by Harlan Sera-lab (Loughborough, UK) and was purified by affinity chromatography with recombinant MBNL1 immobilized on cyanogen bromide-activated Sepharose.
SDS-polyacrylamide gel electrophoresis and Western blotting
HeLa cell extracts were subjected to SDS-PAGE (12.5% acrylamide) as a horizontal strip alongside prestained molecular weight markers (Sigma SDS-7B, Poole, UK) and transferred to nitrocellulose membranes (Schleicher and Schull, Dassel, Germany). After blocking nonspecific sites, membranes containing the HeLa cell extracts were cut into vertical strips and incubated with either primary mAb against MBNL1 (MB1a culture supernatant) or a control hybridoma culture supernatant at a dilution of 1 : 100. After incubation with peroxidase-labelled rabbit anti-mouse Ig (Dako, Ely, UK), specific binding was visualized using a chemiluminescent detection system (SuperSignal, Pierce, Rockford, IL).
Eukaryotic cell culture
Myoblasts from quadriceps muscles of a 15-week DM1 fetus with 1800 CAG repeats were established in culture as described previously (Edom et al. 1994). DM1 and DM2 fibroblasts were established in culture from skin biopsies (Fardaei et al. 2002), following appropriate local ethical regulations and were grown in DMEM with 20% decomplemented fetal bovine serum, 2 mM L-glutamine and antibiotics (Invitrogen, Paisley, UK). All cultures were incubated at 37 °C in a humid air atmosphere containing 5% CO2. After the incubation period, cells on coverslips were fixed with 50 : 50 acetone–methanol for 5 min.
Immunohistochemistry and in situ hybridization
Cells on coverslips were washed 4 times with casein buffer (0.1% casein in 154 mM NaCl and 10 mM Tris, pH 7.6). For double labeling, cells were first incubated with polyclonal antisera for 1 h at 37 ºC, washed 4 times with casein buffer and then incubated with the mAb for 1 h at 37 ºC. Rabbit polyclonal antisera were against MBNL1 (1 : 100), hnRNP-H (Chou et al. 1999: 1 : 100), PSP1 (Fox et al. 2002: 1 : 500) and YT521-B (Nayler et al. 2000: 1 : 500). Mouse mAbs MBNL1 (MB1a), Y12 (Lerner et al. 1981) and 7.13 (Billings et al. 1985) were all diluted 1 : 3, mAb 9G8 (Cavaloc et al. 1994) was diluted 1 : 20 and anti-Y14 mAb (Sigma, Y1253) was diluted 1 : 200. A human autoantiserum against PM/Scl-100 (Brouwer et al. 2001) was diluted 1 : 400. Following incubation with primary antibodies, cells were washed 4 times with casein buffer and incubated with the secondary antibodies diluted in PBS containing 1% horse serum, 1% fetal bovine serum and 0.1% BSA, for 1 h at 37 ºC. Secondary antibody pairs were either 5 µg/mL goat anti-rabbit ALEXA 488 (Molecular Probes, Eugene, OR, USA) and 5 µg/mL goat anti-mouse ALEXA 546 (Molecular Probes) or 10 µg/mL donkey anti-rabbit TRITC (Chemicon, Hampshire, UK) and 20 µg/mL horse anti-mouse FITC (Vector Labs, Burlingame, CA). Human autoantiserum was detected with biotinylated goat anti-human IgG (1 : 100) followed by fluorescein avidin DCS (1 : 100). For dual immunolocalization, mAbs 9G8 and SC35 (S4045; Sigma) were labelled with ALEXA dyes using Zenon mouse IgG1 labeling kit (Z25070, Molecular Probes, Invitrogen, Paisley, UK). Diamidino phenylindole (DAPI) was added for the final 5 min of incubation to counterstain nuclei before mounting in Hydromount (Merck, Nottingham, UK).
Labeling of ribonuclear foci was based on the methods of Taneja et al. (1995) and Fardaei et al. (2001). Cells on coverslips were fixed in 4% paraformaldehyde, 5 mM MgCl2 in PBS for 10 min and washed with 2x SSC (300 mM sodium chloride and 30 mM sodium citrate pH 7). Cells were treated with 40% deionised formamide in 2x SSC for 5 min, which was removed prior to addition of the in situ hybridization mix. The in situ hybridization mixes were prepared in 10% dextran sulphate and 40% formamide in 2x SSC as follows: 0.2% bovine serum albumin, 0.1 mg/mL herring sperm DNA, 0.1 mg/mL baker's yeast transfer RNA (Sigma) and 4 mM ribonucleoside vanadyl complexes (Sigma). The probes used were either 200 nM Cy3-labelled (CAG)10 oligonucleotide (Qiagen, Cologne, Germany) for DM1 or 200 nM Cy3-labelled (CAGG)10 oligonucleotide for DM2. 200-µL of in situ hybridization mix was added to each coverslip. Coverslips were then placed in a humidified chamber and incubated at 37 °C for 16 h. Cells were then washed thoroughly in 2x SSC and mounted in Hydromount.
For combined immunofluorescence and in situ hybridization, all washing steps were done with 2x SSC and antibodies were diluted in 2x SSC. Cells on coverslips were fixed in 50 : 50 acetone–methanol for 5 min and washed. The mAb was diluted in 2x SSC and placed on the coverslips for 1 h at 37 °C. Following washing, 5 µg/mL of goat anti-mouse ALEXA 488 (Molecular Probes) in 2x SSC was added for 1 h at 37 °C. The labelled cells were then treated with 4% paraformaldehyde, 5 mM MgCl2 for 1 min and 40% formamide for 1 min prior to addition of the in situ hybridization mix, as described previously. Cells were examined and sequential confocal scans performed with a Nikon Eclipse E600 epifluorescence microscope with a BioRad MicroRadiance 2000 confocal scanning system (Zeiss, Welwyn Garden City, UK) or a Leica TCS SP5 spectral confocal microscope (Leica Microsystems, Milton Keynes, UK). Analysis of confocal images was performed with IMAGEJ 1.36b software. (Rasband, <http://rsb.info.nih.gov/ij/>, 1997–2006.)
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: glenn.morris{at}rjah.nhs.uk
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References |
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Received: 15 February 2007
Accepted: 6 June 2007
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