the biopsies used in this study were taken , under sterile conditions , from the biceps brachii of four dm2 patients , after informed consent.the histological diagnosis was performed on serial sections processed for routine histological or histochemical staining , based on the clinical criteria set by the international consortium for myotonic dystrophies ( moxley et al . , 2002 ) . the biopsies from donors were trimmed of blood vessels , fat and connective tissues , and rinsed in phosphate - buffered saline ( pbs ) ; satellite cells were isolated as reported in cardani et al . ( 2009 ) and placed in ham 's f10 medium ( sigma - aldrich , buchs , switzerland ) supplemented with 15% fetal bovine serum ( gibco , invitrogen , italy ) , 0.5 mg / ml albumin from bovine serum ( bsa , sigma - aldrich ) , 0.5 mg / ml fetuin , 0.39 g / ml dexamethasone , 10 ng / ml epidermal growth factor , 0.05 mg / ml insulin , 3 mg / ml glucose , 100 u / ml penicillin and 100 g / ml streptomycin ( sigma - aldrich).the myoblasts obtained by this procedure were propagated in plastic flasks at 37c in a humidified 95% air/5% co2 atmosphere . for transmission electron microscopy , the myoblasts were fixed in the flasks and then collected by scraping , whereas for light microscopy they were previously planted on glass coverslips to be processed for fluorescence in situ hybridization ( fish ) and immunocytochemistry ( see below ) . for fluorescence in situ hybridization ( fish ) and immunocytochemistry , myoblasts were fixed with 2% formaldehyde in pbs for 30 min at 4c . for fish , a texas red labelled ( cagg)5 probe ( idt , coralville , ia , usa ) was used as previously reported by cardani et al . briefly , fixed myoblasts were permeabilized with 2% acetone in pbs ( pre - chilled at 20c ) for 5 min . after washing in pbs , sections were incubated in 30% formamide and 2ssc for 10 min at room temperature ( r.t . ) , and then hybridized with the probe ( 1 ng/l ) for 2 h at 37c in 30% formamide , 2ssc , 0.02% bsa , 67 ng/l yeast trna , 2 mm vanadyl ribonucleoside complex . cells were washed in 30% formamide and 2ssc at 45c for 30 min , washed five times in pbs for 3 min at r.t . , and pre - incubated with normal goat serum ( dako , glostrup , denmark ) at a dilution 1:20 in pbs containing 2% bsa for 20 min at r.t .. immunolabelling for mbnl1 protein was performed using a rabbit polyclonal anti - mbnl1 ( kind gift of prof . c. a. thornton , university of rochester , ny , usa ) at a dilution of 1:1000 in pbs containing 2% bsa , for 2 hr at r.t . , then revealed with an alexa 488-labelled goat anti - rabbit antibody ( molecular probes , invitrogen , italy ) diluted 1:200 in pbs containing 2% bsa for 1 h at r.t .. after incubation , the cells were stained for dna with 165 nm 4.6 diamidino-2-phenylindole ( dapi ) in pbs at r.t . for 30 min , and to get information on the intranuclear location of the rna - containing foci and their composition , a panel of antibodies directed against mbnl1 or transcription , splicing or cleavage factors were used in dual - immunolabelling experiments ( table 1 ) ; secondary antibodies labelled with different fluorochromes were used . briefly , 2% formaldehyde - fixed myoblasts were post - fixed with cold 70% ethanol , and incubated with the primary and the appropriate secondary antibodies ( for 2 h and 1 h respectively , at r.t . ) ; immunolabelled myoblasts were stained for dna with 0.1 g / ml of hoechst 33258 in pbs ( 10 min at r.t . ) , and finally mounted in a drop of mowiol ( calbiochem , milan , italy ) . table 1features of the antibodies used for immunofluorescence analyses.antibodyoriginantigendilutionsecondary markerreference - sourceanti - c8vchicken polyclonalhnrnp core proteins1:1000alexa 594 labelled goat anti - chicken iggjones et al . , 1980anti - smmouse monoclonalsm antigen ( u1 , u2 , u4/6 , u5 snrnp)1:100alexa 594 labelled goat anti - mouse iggabcam ( cambridge , uk)anti - sc35mouse monoclonalsc-35 non snrnp splicing factor1:200alexa 594 labelled goat anti - mouse iggsigma chem co. ( st . louis mo , usa)anti - cstfchicken polyclonalcleavage stimulation factor1:2000alexa 594 labelled goat anti - chicken iggveraldi et al . , 2001anti - rna polymerase ii ( clone h14)mouse monoclonalphosphorylated ( activated ) form of rna polymerase ii1:100alexa 594 labelled goat anti - mouse igmresearch diagnostics inc . ( concorde ma , usa)anti - pmlmouse monoclonalhuman pml protein1:200alexa 594 labelled goat anti - mouse iggsanta cruz biotechnology ( santa cruz ca , usa ) as negative controls , some slides were processed as described above , but omitting the incubation with the primary antibodies . an olympus bx51 microscope equipped with a 100w mercury lamp was used under the following conditions : 330385 nm excitation filter ( excf ) , 400-nm dichroic mirror ( dm ) , and 420-nm barrier filter ( bf ) , for hoechst 33258 or dapi ; 450480 nm excf , 500-nm dm , and 535 nm20 nm bandpass filter , for the fluorescence of alexa 488 ; 540 nm excf , 580 nm dm , and 620 nm bf for alexa 594 or texas red . images were recorded with an olympus magnifire camera system , and processed with the olympus cellf software ( olympus italia srl , segrate , italy ) . for conventional morphology , cells were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 m srensen phosphate buffer at 4c for 1 h , washed , post - fixed with 1% oso4 at 4c for 30 min , dehydrated with acetone and embedded in epon . for immunoelectron microscopy , cells were fixed with a mixture of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 m srensen phosphate buffer at 4c for 1 h , washed , treated with nh4cl 0.5 m in pbs , dehydrated with ethanol and embedded in unicryl resin . ultrathin sections from epon - embedded samples were conventionally stained with uranyl acetate and lead citrate . ultrathin sections from unicryl - embedded samples were placed on nickel grids and processed for immunocytochemistry with the same anti - mbnl1 antibody used for fluorescence microscopy . 1:100 in pbs and incubated overnight at 4c with the primary antibody diluted 1:50 in a solution containing 0.1% bsa and 0.05% tween 20 in pbs . after rinsing , sections were floated in ngs and then reacted for 20 min with the specific gold - conjugated secondary antibody ( jackson immunoresearch , west grove , pa ) diluted 1:10 in pbs . finally , the sections were rinsed and air dried . as controls , some grids were treated with the incubation mixture without the primary antibody and then processed as described above . moreover , in order to confirm the apparent co - location of mbnl1 with some splicing factors as observed by immunofluorescence microscopy ( see the section results ) , myoblast samples were also dual - immunolabelled by combining the anti - mbnl1 antibody with antibodies directed against either the sm complex of u1 , u2 , u5 and u4/u6 snrnps ( abcam , cambridge , ma ) or the heterogeneous nuclear rnp ( hnrnp ) core protein ( jones et al . , 1980 ) , according to the procedure described above . to reduce chromatin contrast and selectively reveal nuclear rnp constituents , the sections were treated according to the edta method ( bernhard , 1969 ) , and then observed in a philips morgagni tem operating at 80kv and equipped with a megaview ii camera for digital image acquisition . the biopsies used in this study were taken , under sterile conditions , from the biceps brachii of four dm2 patients , after informed consent.the histological diagnosis was performed on serial sections processed for routine histological or histochemical staining , based on the clinical criteria set by the international consortium for myotonic dystrophies ( moxley et al . , 2002 ) . the biopsies from donors were trimmed of blood vessels , fat and connective tissues , and rinsed in phosphate - buffered saline ( pbs ) ; satellite cells were isolated as reported in cardani et al . ( 2009 ) and placed in ham 's f10 medium ( sigma - aldrich , buchs , switzerland ) supplemented with 15% fetal bovine serum ( gibco , invitrogen , italy ) , 0.5 mg / ml albumin from bovine serum ( bsa , sigma - aldrich ) , 0.5 mg / ml fetuin , 0.39 g / ml dexamethasone , 10 ng / ml epidermal growth factor , 0.05 mg / ml insulin , 3 mg / ml glucose , 100 u / ml penicillin and 100 g / ml streptomycin ( sigma - aldrich).the myoblasts obtained by this procedure were propagated in plastic flasks at 37c in a humidified 95% air/5% co2 atmosphere . for transmission electron microscopy , the myoblasts were fixed in the flasks and then collected by scraping , whereas for light microscopy they were previously planted on glass coverslips to be processed for fluorescence in situ hybridization ( fish ) and immunocytochemistry ( see below ) . for fluorescence in situ hybridization ( fish ) and immunocytochemistry , myoblasts were fixed with 2% formaldehyde in pbs for 30 min at 4c . for fish , a texas red labelled ( cagg)5 probe ( idt , coralville , ia , usa ) was used as previously reported by cardani et al . briefly , fixed myoblasts were permeabilized with 2% acetone in pbs ( pre - chilled at 20c ) for 5 min . after washing in pbs , sections were incubated in 30% formamide and 2ssc for 10 min at room temperature ( r.t . ) , and then hybridized with the probe ( 1 ng/l ) for 2 h at 37c in 30% formamide , 2ssc , 0.02% bsa , 67 ng/l yeast trna , 2 mm vanadyl ribonucleoside complex . cells were washed in 30% formamide and 2ssc at 45c for 30 min , washed five times in pbs for 3 min at r.t . , and pre - incubated with normal goat serum ( dako , glostrup , denmark ) at a dilution 1:20 in pbs containing 2% bsa for 20 min at r.t .. immunolabelling for mbnl1 protein was performed using a rabbit polyclonal anti - mbnl1 ( kind gift of prof . c. a. thornton , university of rochester , ny , usa ) at a dilution of 1:1000 in pbs containing 2% bsa , for 2 hr at r.t . , then revealed with an alexa 488-labelled goat anti - rabbit antibody ( molecular probes , invitrogen , italy ) diluted 1:200 in pbs containing 2% bsa for 1 h at r.t .. after incubation , the cells were stained for dna with 165 nm 4.6 diamidino-2-phenylindole ( dapi ) in pbs at r.t . for 30 min , and to get information on the intranuclear location of the rna - containing foci and their composition , a panel of antibodies directed against mbnl1 or transcription , splicing or cleavage factors were used in dual - immunolabelling experiments ( table 1 ) ; secondary antibodies labelled with different fluorochromes were used . briefly , 2% formaldehyde - fixed myoblasts were post - fixed with cold 70% ethanol , and incubated with the primary and the appropriate secondary antibodies ( for 2 h and 1 h respectively , at r.t . ) ; immunolabelled myoblasts were stained for dna with 0.1 g / ml of hoechst 33258 in pbs ( 10 min at r.t . ) , and finally mounted in a drop of mowiol ( calbiochem , milan , italy ) . table 1features of the antibodies used for immunofluorescence analyses.antibodyoriginantigendilutionsecondary markerreference - sourceanti - c8vchicken polyclonalhnrnp core proteins1:1000alexa 594 labelled goat anti - chicken iggjones et al . , 1980anti - smmouse monoclonalsm antigen ( u1 , u2 , u4/6 , u5 snrnp)1:100alexa 594 labelled goat anti - mouse iggabcam ( cambridge , uk)anti - sc35mouse monoclonalsc-35 non snrnp splicing factor1:200alexa 594 labelled goat anti - mouse iggsigma chem co. ( st . louis mo , usa)anti - cstfchicken polyclonalcleavage stimulation factor1:2000alexa 594 labelled goat anti - chicken iggveraldi et al . , 2001anti - rna polymerase ii ( clone h14)mouse monoclonalphosphorylated ( activated ) form of rna polymerase ii1:100alexa 594 labelled goat anti - mouse igmresearch diagnostics inc . ( concorde ma , usa)anti - pmlmouse monoclonalhuman pml protein1:200alexa 594 labelled goat anti - mouse iggsanta cruz biotechnology ( santa cruz ca , usa ) as negative controls , some slides were processed as described above , but omitting the incubation with the primary antibodies . an olympus bx51 microscope equipped with a 100w mercury lamp was used under the following conditions : 330385 nm excitation filter ( excf ) , 400-nm dichroic mirror ( dm ) , and 420-nm barrier filter ( bf ) , for hoechst 33258 or dapi ; 450480 nm excf , 500-nm dm , and 535 nm20 nm bandpass filter , for the fluorescence of alexa 488 ; 540 nm excf , 580 nm dm , and 620 nm bf for alexa 594 or texas red . images were recorded with an olympus magnifire camera system , and processed with the olympus cellf software ( olympus italia srl , segrate , italy ) . for conventional morphology , cells were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 m srensen phosphate buffer at 4c for 1 h , washed , post - fixed with 1% oso4 at 4c for 30 min , dehydrated with acetone and embedded in epon . for immunoelectron microscopy , cells were fixed with a mixture of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 m srensen phosphate buffer at 4c for 1 h , washed , treated with nh4cl 0.5 m in pbs , dehydrated with ethanol and embedded in unicryl resin . ultrathin sections from epon - embedded samples were conventionally stained with uranyl acetate and lead citrate . ultrathin sections from unicryl - embedded samples were placed on nickel grids and processed for immunocytochemistry with the same anti - mbnl1 antibody used for fluorescence microscopy . sections were floated on normal goat serum ( ngs ) diluted 1:100 in pbs and incubated overnight at 4c with the primary antibody diluted 1:50 in a solution containing 0.1% bsa and 0.05% tween 20 in pbs . after rinsing , sections were floated in ngs and then reacted for 20 min with the specific gold - conjugated secondary antibody ( jackson immunoresearch , west grove , pa ) diluted 1:10 in pbs . finally , the sections were rinsed and air dried . as controls , some grids were treated with the incubation mixture without the primary antibody and then processed as described above . moreover , in order to confirm the apparent co - location of mbnl1 with some splicing factors as observed by immunofluorescence microscopy ( see the section results ) , myoblast samples were also dual - immunolabelled by combining the anti - mbnl1 antibody with antibodies directed against either the sm complex of u1 , u2 , u5 and u4/u6 snrnps ( abcam , cambridge , ma ) or the heterogeneous nuclear rnp ( hnrnp ) core protein ( jones et al . , 1980 ) , according to the procedure described above . to reduce chromatin contrast and selectively reveal nuclear rnp constituents , the sections were treated according to the edta method ( bernhard , 1969 ) , and then observed in a philips morgagni tem operating at 80kv and equipped with a megaview ii camera for digital image acquisition . in dm2 myoblasts , mbnl1 proved to be present in discrete dots ( which correspond to the expanded - ccug - containing foci : figure 1 ) as well as in a weakly fluoresceing diffused form throughout the nucleoplasm ( consistent with the intranuclear location of splicing factors ) . dual immunolabelling experiments demonstrated that mbnl1 does not co - locate in the foci with the non - snrnp splicing factor sc35 ( figure 2 ) and the pml protein ( figure 3 ) ; similar results were obtained also for rna polymerase ii and the cleavage factor cstf ( not shown ) . figure 1 in the myoblast nuclei , the immunolabelling for mbn1 ( green fluorescence in a ) and the signal of in situ hybridization for the mutant mrna containing ccug repeats ( red fluorescence in b ) co - locate ( yellow fluorescence in the merged image , c ) . bar : 10 m . 
  in the myoblast nuclei , the immunolabelling for mbn1 ( green fluorescence in a ) and the signal of in situ hybridization for the mutant mrna containing ccug repeats ( red fluorescence in b ) co - locate ( yellow fluorescence in the merged image , c ) . figure 2the immunolabelling for mbnl1 ( green fluorescence in a ) and that for the non - snrnp splicing factor sc35 ( red fluorescence in b ) do not co - locate ( merged image , c ) . the immunolabelling for mbnl1 ( green fluorescence in a ) and that for the non - snrnp splicing factor sc35 ( red fluorescence in b ) do not co - locate ( merged image , c ) . figure 3the immunolabelling for mbnl1 ( green fluorescence in a ) and that for the pml protein ( red fluorescence in b ) do not co - locate ( merged image , c ) . the immunolabelling for mbnl1 ( green fluorescence in a ) and that for the pml protein ( red fluorescence in b ) do not co - locate ( merged image , c ) . the hnrnp core protein ( figure 4 ) and the snrnp sm antigen ( figure 5 ) were observed in some of the mbnl1-positive foci . interestingly enough , the immunolabelling for these rnps appeared as a ring - shaped fluorescence pattern around the brightly fluoresceing mbnl1-contaning core ( insets of figure 4 and 5 ) . in addition , some co - location for the hnrnp core protein and for the sm antigen was also observed to occur also in the nucleoplasm ( figure 4 and 5 ) . figure 4the immunolabelling for mbnl1 ( green fluorescence in a ) and that for the hnrnp core protein ( red fluorescence in b ) co - locate ( yellow fluorescence in the merged image , c ) . bar : 10 m . in the insets , the co - labelled focus indicated by the arrow the immunolabelling for mbnl1 ( green fluorescence in a ) and that for the hnrnp core protein ( red fluorescence in b ) co - locate ( yellow fluorescence in the merged image , c ) . bar : 10 m . in the insets , the co - labelled focus indicated by the arrow figure 5the immunolabelling for mbnl1 ( green fluorescence in a ) and that for the snrnp sm antigen ( red fluorescence in b ) co - locate ( yellow fluorescence in the merged image , c ) . bar : 10 m . in the insets , the co - labelled focus indicated by the arrow is shown at a higher magnification . the immunolabelling for mbnl1 ( green fluorescence in a ) and that for the snrnp sm antigen ( red fluorescence in b ) co - locate ( yellow fluorescence in the merged image , c ) bar : 10 m . in the insets , the co - labelled focus indicated by the arrow fluorescent labelling was negligible in the negative controls ( not shown ) . at electron microscopy , myoblast nuclei showed small condensed chromatin clumps ( mainly at the nuclear periphery ) , pg and abundant pf preferentially located at the hedge of condensed chromatin , and small clusters of ig in the interchromatin space ; the nucleoli were large with a few fibrillar centres surrounded by the dense fibrillar component and a prominent granular component ( figure 6 ) . in addition to the usual structural constituents , roundish nuclear domains ( 100200 nm in diameter ) were observed in about 20% of the sectioned nuclei : in epon - embedded samples they appeared as roundish homogeneously strong electron - dense structures ( figure 4 ) , while in the unicryl - embedded and edta - treated samples they appeared as less electron - dense irregularly sized domains surrounded by fine fibrils spreading out at their periphery ( figure 7 ) . the gold - immunolabelling for mbnl1 specifically occurred on pf and was found to accumulate in the nuclear domains described above ( figure 7a ) . consistent with the evidence in fluorescence microscopy , mbnl1 was observed to co - locate in the foci with hnrnps ( figure 7b ) and with the snrnp sm antigen ( figure 7c ) . on the thin sections , however , the gold grains labelling these rnps were distributed all over the foci and not at the periphery only . figure 6myoblast nucleus , glutaraldehyde fixation , oso4 postfixation , epon embedding . a strongly electron - dense roundish nuclear domain ( arrow ) myoblast nucleus , glutaraldehyde fixation , oso4 postfixation , epon embedding . a strongly electron - dense roundish nuclear domain ( arrow ) ( a ) immunolabelling with anti - mbnl1 antibody : the signal accumulates in the rnp nuclear domain ( arrows ) and is also associated with pf ( arrowheads ) . ( b ) dual immunolabelling with anti - mbnl1 ( 12 nm ) and anti - snrnp ( 6 nm ) antibodies ; ( c ) dual immunolabelling with anti - mbnl1 ( 18 nm ) and anti - hnrnp ( 12 nm ) antibodies : in both cases the probes co - locate in the rnp nuclear domain ( arrow ) and on pf ( arrowheads ) . ( a ) immunolabelling with anti - mbnl1 antibody : the signal accumulates in the rnp nuclear domain ( arrows ) and is also associated with pf ( arrowheads ) . ( b ) dual immunolabelling with anti - mbnl1 ( 12 nm ) and anti - snrnp ( 6 nm ) antibodies ; ( c ) dual immunolabelling with anti - mbnl1 ( 18 nm ) and anti - hnrnp ( 12 nm ) antibodies : in both cases the probes co - locate in the rnp nuclear domain ( arrow ) and on pf ( arrowheads ) . bars : 250 nm ( a  c ) ; 100 nm ( insets ) . in dm2 myoblasts , mbnl1 proved to be present in discrete dots ( which correspond to the expanded - ccug - containing foci : figure 1 ) as well as in a weakly fluoresceing diffused form throughout the nucleoplasm ( consistent with the intranuclear location of splicing factors ) . dual immunolabelling experiments demonstrated that mbnl1 does not co - locate in the foci with the non - snrnp splicing factor sc35 ( figure 2 ) and the pml protein ( figure 3 ) ; similar results were obtained also for rna polymerase ii and the cleavage factor cstf ( not shown ) . figure 1 in the myoblast nuclei , the immunolabelling for mbn1 ( green fluorescence in a ) and the signal of in situ hybridization for the mutant mrna containing ccug repeats ( red fluorescence in b ) co - locate ( yellow fluorescence in the merged image , c ) . bar : 10 m . 
  in the myoblast nuclei , the immunolabelling for mbn1 ( green fluorescence in a ) and the signal of in situ hybridization for the mutant mrna containing ccug repeats ( red fluorescence in b ) co - locate ( yellow fluorescence in the merged image , c ) . figure 2the immunolabelling for mbnl1 ( green fluorescence in a ) and that for the non - snrnp splicing factor sc35 ( red fluorescence in b ) do not co - locate ( merged image , c ) . the immunolabelling for mbnl1 ( green fluorescence in a ) and that for the non - snrnp splicing factor sc35 ( red fluorescence in b ) do not co - locate ( merged image , c ) . figure 3the immunolabelling for mbnl1 ( green fluorescence in a ) and that for the pml protein ( red fluorescence in b ) do not co - locate ( merged image , c ) . the immunolabelling for mbnl1 ( green fluorescence in a ) and that for the pml protein ( red fluorescence in b ) do not co - locate ( merged image , c ) . the hnrnp core protein ( figure 4 ) and the snrnp sm antigen ( figure 5 ) were observed in some of the mbnl1-positive foci . interestingly enough , the immunolabelling for these rnps appeared as a ring - shaped fluorescence pattern around the brightly fluoresceing mbnl1-contaning core ( insets of figure 4 and 5 ) . in addition , some co - location for the hnrnp core protein and for the sm antigen was also observed to occur also in the nucleoplasm ( figure 4 and 5 ) . figure 4the immunolabelling for mbnl1 ( green fluorescence in a ) and that for the hnrnp core protein ( red fluorescence in b ) co - locate ( yellow fluorescence in the merged image , c ) . bar : 10 m . in the insets , the co - labelled focus indicated by the arrow the immunolabelling for mbnl1 ( green fluorescence in a ) and that for the hnrnp core protein ( red fluorescence in b ) co - locate ( yellow fluorescence in the merged image , c ) . bar : 10 m . in the insets , the co - labelled focus indicated by the arrow figure 5the immunolabelling for mbnl1 ( green fluorescence in a ) and that for the snrnp sm antigen ( red fluorescence in b ) co - locate ( yellow fluorescence in the merged image , c ) . bar : 10 m . in the insets , the co - labelled focus indicated by the arrow is shown at a higher magnification . the immunolabelling for mbnl1 ( green fluorescence in a ) and that for the snrnp sm antigen ( red fluorescence in b ) co - locate ( yellow fluorescence in the merged image , c ) bar : 10 m . in the insets , the co - labelled focus indicated by the arrow at electron microscopy , myoblast nuclei showed small condensed chromatin clumps ( mainly at the nuclear periphery ) , pg and abundant pf preferentially located at the hedge of condensed chromatin , and small clusters of ig in the interchromatin space ; the nucleoli were large with a few fibrillar centres surrounded by the dense fibrillar component and a prominent granular component ( figure 6 ) . in addition to the usual structural constituents , roundish nuclear domains ( 100200 nm in diameter ) were observed in about 20% of the sectioned nuclei : in epon - embedded samples they appeared as roundish homogeneously strong electron - dense structures ( figure 4 ) , while in the unicryl - embedded and edta - treated samples they appeared as less electron - dense irregularly sized domains surrounded by fine fibrils spreading out at their periphery ( figure 7 ) . the gold - immunolabelling for mbnl1 specifically occurred on pf and was found to accumulate in the nuclear domains described above ( figure 7a ) . consistent with the evidence in fluorescence microscopy , mbnl1 was observed to co - locate in the foci with hnrnps ( figure 7b ) and with the snrnp sm antigen ( figure 7c ) . on the thin sections , however , the gold grains labelling these rnps were distributed all over the foci and not at the periphery only . figure 6myoblast nucleus , glutaraldehyde fixation , oso4 postfixation , epon embedding . a strongly electron - dense roundish nuclear domain ( arrow ) a strongly electron - dense roundish nuclear domain ( arrow ) occurs in the nucleoplasm . bar : 1 m . 
 figure 7myoblast nuclei , paraformaldehyde fixation , unicryl - embedding , edta staining . ( a ) immunolabelling with anti - mbnl1 antibody : the signal accumulates in the rnp nuclear domain ( arrows ) and is also associated with pf ( arrowheads ) . ( b ) dual immunolabelling with anti - mbnl1 ( 12 nm ) and anti - snrnp ( 6 nm ) antibodies ; ( c ) dual immunolabelling with anti - mbnl1 ( 18 nm ) and anti - hnrnp ( 12 nm ) antibodies : in both cases the probes co - locate in the rnp nuclear domain ( arrow ) and on pf ( arrowheads ) . bars : 250 nm ( a  c ) ; 100 nm ( insets ) . 
  myoblast nuclei , paraformaldehyde fixation , unicryl - embedding , edta staining . ( a ) immunolabelling with anti - mbnl1 antibody : the signal accumulates in the rnp nuclear domain ( arrows ) and is also associated with pf ( arrowheads ) . ( b ) dual immunolabelling with anti - mbnl1 ( 12 nm ) and anti - snrnp ( 6 nm ) antibodies ; ( c ) dual immunolabelling with anti - mbnl1 ( 18 nm ) and anti - hnrnp ( 12 nm ) antibodies : in both cases the probes co - locate in the rnp nuclear domain ( arrow ) and on pf ( arrowheads ) . bars : 250 nm ( a  c ) ; 100 nm ( insets ) . combining fluorescence and immunoelectron microscopy on dm2 cultured myoblasts , we obtained information on the molecular composition of the typical nuclear foci were the expanded - ccug - containing transcripts accumulate ; in addition these foci were recognized as distinct nuclear domains at the ultrastructural level . experiments of simultaneous in situ hybridization and immunolabelling confirmed that in cultured myoblasts the mutant rna - containing nuclear foci sequester the splicing factor mbnl1 ( cardani et al . , 2009 ) , thus indicating that the molecular features observed in dm2 adult tissues are reproduced in in vitro muscle cells ( mankodi et al . the rna - containing nuclear foci observed at fluorescence microscopy can be also recognized at electron microscopy as roundish electron - dense domains which do contain rnps ( as demonstrated by the edta regressive staining ) , and proved to be immunopositive for mbnl1 . these nuclear domains are always surrounded by pf but never occur in proximity of ig clusters , consistent with previous observations by immunofluorescence techniques ( holt et al . , 2007 ) . they morphologically resemble the so - called amorphous bodies , i.e. , rnp - containing nuclear aggregates which accumulate in several vertebrate tissues during hibernation , and are storage sites for many protein factors involved in pre - mrna processing ( e.g. , malatesta et al . the diffuse immunolabelling for mbnl1 observed in myoblast nuclei at fluorescence microscopy was found to correspond to the ultrastructural labelling of pf , where transcription and early splicing of pre - mrna take place ( fakan , 2004 ) ; this distribution is in agreement with the functional role of mbnl1 as an rna - binding protein involved in the regulation of alternative splicing ( pascual et al . , 2006 ) . fluorescence dual - immunolabelling experiments demonstrated that rna polymerase ii , the non - snrnp splicing factor sc35 , the cleavage factor cstf , and the pml protein do not co - locate with mbnl1 in the foci , whereas a co - occurrence herein was observed for the hnrnp core protein and the snrnp sm antigen . this co - location was confirmed by immunoelectron microscopy not only on the rnp - containing nuclear domains but also on pf ; this latter finding corresponds to the diffuse co - location observed at light microscopy . interestingly , in electron micrographs the labelling for hnrnps and snrnps was distributed over the whole nuclear domains , whereas at fluorescence microscopy a bright ring - shaped signal for hnrnps or snrnps was found to surround the mbnl1-rich core of the foci . to explain this inconsistence , one should consider that at electron microscopy the antibodies can interact with their antigens even inside the foci , since the immunolabelling is performed on sections ; on the contrary , whole cells are used for the immunofluorescent labelling , so that the penetration of the probe could be hindered by the apparently dense structure of the rnp - containing foci . the intense fluorescence for hnrnps and snrnps around the foci could also be explained by the high content in these two splicing factors on pf which surround the mbnl1-containing core . the presence of hnrnps and snrnps indicates that the dm2-specific nuclear foci share similarities in their molecular composition with both the amorphous bodies and the herds , i.e. , the heterogeneous aggregates containing at least two nuclear rna - associated protein factors which usually do not co - locate . amorphous bodies form during hibernation , when the transcription and splicing rate is drastically reduced , and disassemble upon arousal , releasing the pre - mrna processing factors accumulated therein , in order to rapidly resume the nuclear activities ( malatesta et al . , 2001 ) . herds were observed in different cell systems , whenever rna transcription is altered , and have been suggested as a possible marker of transcriptional arrest ( biggiogera and pellicciari , 2000 ) : under normal conditions , a balance exists between the amount of newly synthesized pre - mrna and the proteins involved in their processing but , if the amount of newly formed hnrna decreases , rna - associated proteins become exceedingly predominant and accumulate in herds . the present results suggest that an exceeding amount of rna ( i.e. , the expanded - ccug - containing transcripts retained in the nucleus of dm2 cells ) can determine the sequestration in the foci not only of mbnl1 but also of other pre - mrna processing factors . some of the pathological features of dm are thought to be caused by the mis - regulated alternative splicing of rna due , at least in part , to the mis - localization of mbnl1 ( review in osborne and thornton , 2006 ) . the sequestration of other splicing factors involved in early phases of pre - mrna processing demonstrated by the present study strengthens the idea of a general alteration in the maturation of several mrnas , which could lead to the multiple pathological dysfunctions observed in dystrophic patients.the occurrence of hnrnps and snrnps in some ( but not all the ) rna - containing foci suggests that a possible limiting factor for their sequestration could be the amount of ccug repeats and/or mbnl1 accumulated therein ; taking into account that the severity of the dm2 phenotype relates to the amplification of tetraplet repeats , this opens interesting perspectives for future studies .
myotonic dystrophy type 2 ( dm2 ) is a dominantly inherited autosomal disease with multi - systemic clinical features and it is caused by expansion of a cctg tetranucleotide repeat in the first intron of the zinc finger protein 9 ( znf9 ) gene in 3q21.the expanded - ccug - containing transcripts are retained in the cell nucleus and accumulate in the form of focal aggregates which specifically sequester the muscleblind - like 1 ( mbnl1 ) protein , a rna binding factor involved in the regulation of alternative splicing . 
 the structural organization and composition of the foci are still incompletely known . in this study , the nuclear foci occurring in cultured myoblasts from dm2 patients were characterised at fluorescence and transmission electron microscopy by using a panel of antibodies recognizing transcription and processing factors of pre - mrnas . 
 mbnl1 proved to co - locate in the nuclear foci with snrnps and hnrnps , whereas no co - location was observed with rna polymerase ii , the non - rnp splicing factor sc35 , the cleavage factor cstf and the pml protein . at electron 
 microscopy the mbnl1-containing nuclear foci appeared as roundish domains showing a rather homogeneous structure and proved to contain snrnps and hnrnps . 
 the sequestration of splicing factors involved in early phases of pre - mrna processing supports the hypothesis of a general alteration in the maturation of several mrnas , which could lead to the multiple pathological dysfunctions observed in dystrophic patients .