Histochemis
Histochemistry (1990) 93:497-499
© Springer-Verlag 1;990t~
Muscle fiber typing in routinely processed skeletal muscle with monoclonal antibodies M.G. Havenith, R. Visser, J.M.C.
Schrijvers-van Schendel, and F.T. Bosman*
Department of Pathology, University of Limburg, P.O. Box 616, NL-6200 MD Maastricht, The Netherlands Accepted December 2, 1989
Muscle fiber typing is conventionally performed using mATPase enzyme histochemistry on cryostat sections. After pre-incubation of sections at p H 4.3, 4.6 and 10.3, based on the pattern of enzyme reactivity, the fibers can be classified in types I, II (subtypes A, AB and B) and the intermediate C (I and II) fibers. We have attempted to perform fiber typing of human psoas muscle by immunohistochemistry, using monoclonal antibodies R11D10 (specific for cardiac and type I skeletal myosin) and MY-32 (specific for fast muscle fibers) on cryostat as well as on paraffin sections. Staining of consecutive cryostat sections showed that type I fibers are R11D10 reactive whereas type II fibers are MY-32 reactive. Subtyping of type II fibers could not be performed by immunohistochemistry. Quantitative analysis of type I and II fibers showed that enzyme histochemical and immunohistochemical analysis are in close agreement. Summary.
Introduction
Analysis of myofibrillar ATPase (mATPase) activity after preincubation at different p H is the commonly used method for muscle fiber typing (Brooke and Kaiser 1970), However, this technique has to be applied on serial cryostat sections of fresh frozen tissue. The observed differences in mATPase staining intensity between different fibers correlate with the myosin heavy chain (MHC) content, as demonstrated by electrophoretic analysis of single muscle fibers (Staron and Pette 1986). If specific antibodies to M H C were available these could be used for muscle fiber typing by immunohistochemistry. Along these lines, Gr6schel-Stewart et al. (1973) developed antibodies against myosin from human pectoralis muscle, but these antibodies did not allow an unambiguous differentiation between type I and type II fibers. Billeter et al. (1980), however, succeeded in discriminating h u m a n type I and II fibers with polyclonal antibodies to rabbit slow and fast myosin, which had been raised in guinea pigs. Moore et al. (1984) raised murine monoclonal antibodies specific for type I and type II myosin. If the myosin heavy chains remain immunoreactive subsequent to tissue fixation and paraffin embedding, in principle muscle fiber typing can be performed on paraffin sections, which would obviate * To whom offprint requests should be sent
the need for the more complex procedure o f enzyme cytochemistry on frozen sections. Hitherto three reports dealing with muscle fiber typing in paraffin sections have been published. In these studies a monoclonal antibody (F7) against an insoluble fraction of h u m a n brain stem, with an unidentified antigen, and a monoclonal antibody against neurofilament were used (Dodson et al. 1987; N a k a m u r a et al. 1987; Oldfors et al. 1989). In the present report we describe the application o f the monoclonal antimyosin antibodies R l l D I 0 (Khaw et al. 1984) and MY-32 on frozen and paraffin sections for immunohistochemical fiber typing and a comparison with enzyme histochemical muscle fiber typing with the classical mATPase method. Materials and methods
Human psoas muscle specimens were obtained at autopsy (not more than 2 h after death) and frozen in isopentane quenched in liquid nitrogen. Frozen sections of 12 gm thickness were used for enzymehistochemistry and of 4 gm for immunohistochemistry. Thereafter, the remaining frozen tissue was fixed in neutral buffered paraformaldehyde, embedded in paraffin, and sectioned at 4 gm for immunohistochemistry. The mATPase staining reactions were performed at pH 9.4 following preincubation at pH 4.3, 4.6, and 10.4 respectively (Brooke and Kaiser 1970). The mATPase reactivity after preincubation at different pH for the various fiber types, which is related to the MHC phenotype, is listed in Table l. RlID10 (Khaw et al. 1984) was a kind gift from Centocor Europe (Leiden, The Netherlands). For production of the antibody human cardiac myosin was used as immunogen. R l t D I 0 is of the IgG2a subclass and reacts Table 1. Enzyme histochemically determined mATPase activity and
MHC immunoreactivity of human psoas muscle Muscle fiber mATPase typeS pH 4.6 pH 4.3 I IIA
IIAB IIB
RI1D10 MY-32 pH 10.4
+++ --
+++ -+
--
+++
--
+ ++ + ++
-
++ ++
-
+ +
+ + +
-
+ +
Legends: + + + strong reactivity; + + intermediate reactivity; + weak reactivity; - no reactivity. a fiber type assigned according to Staron and Pette (1986, 1987)
498
Fig. 1 A-E. Serial frozen sections of human psoas muscle. A mATPase staining, pH 4.3, B mATPase staining pH, 4.6, C immunoperoxidase staining with RllD10, D mATPase staining pH 10.4, E immunoperoxidase staining with MY-32. × 235
specifically with cardiac myosin and slow (type I) skeletal myosin. MY-32 (Sigma) is a mouse monoclonal antibody of the IgG1 subclass raised against rabbit skeletal muscle. The antibody reacts specifically with fast (type II) myosin. Both monoclonal antibodies show extensive species cross-reactivity. Frozen sections were fixed in acetone at - 2 0 ° C for 20 min. Paraffin sections were deparaffinized and rehydrated. In both, endogenous peroxidase activity was blocked in 0.3 % HzO2 in methanol for 20 min at room temperature (RT). After washing with phosphate buffered saline (PBS) (3 × 5 min) the sections were incubated with the monoclonal antibodies in a moist chamber at RT. To restore the immunoreactivity for R l l D t 0 and MY-32 in paraffin sections, preincubation with 0.1% pepsin (Sigma) in 0.1 N HCI (30 min at RT) was required. R11DI0 was diluted 1:10000 and MY-32 1 : 4000, both in PBS with 1% bovine serum albumin (BSA). After 3 washes in PBS, peroxidase labeled rabbit anti mouse antibodies, diluted 1:200 in PBS with 1% BSA, were applied for 1 h. After final washing, the immunoreactivity was visualized by a diaminobenzidine-H202 substrate with addition of 0.01 M imidazole. In order to compare the overall results of fiber typing by enzyme histochemistry with those of immunohistochemistry on frozen as well as paraffin sections, fiber types of consecutive fibers were visually classified and expressed as a percentage of all fibers.
(I and II) C fibers intermediate between types I and IIA, was almost absent. Therefore the possibility to identify these intermediate fibers by immunohistochemistry was not explored further. On psoas muscle preparations, fiber typing (in types I and II) was performed in parallel sections using enzyme histochemistry on cryostate sections and immunohistochemistry on cryostat and paraffin sections. Reproducible labeling patterns on immunostained sections were obtained with antibodies R11D10 and MY-32 (Fig. 2). The quantitative d a t a on fiber typing, summarized in Table 2, demonstrate that identical results are obtained using these methods.
Results By m A T P a s e enzyme histochemistry on cryostat sections o f h u m a n psoas muscle after preincubation at p H 4.3 and p H 4.6 the usual mosaic pattern was obtained with intensely stained, weakly stained and unstained fibers (Fig. 1 A and 1 B). Intensely staining fibers showed strong reactivity with R l l D 1 0 and no reactivity with MY-32 in parallel frozen sections (Fig. 1 C). These fibers can be classified as type I. Unstained fibers, in contrast, showed intense immunoreactivity with MY-32 (Fig. 1 E). These fibers can be classified as type II. By immunohistochemistry on the basis o f MY-32 reactivity these fibers could not be further subclassified. By enzyme histochemistry subclassification of type II fibers could be performed according to the staining intensity at p H 4.6 and 10.4. In the psoas muscle intermediate m A T P a s e reactivity at p H 4.6, which would identify the
Fig. 2A, B. Serial paraffin sections of human psoas muscle. A immunoperoxidase staining with RllD10, B immunoperoxidase staining with MY-32. × 235
499 Table 2. Quantitative assessment of fiber types using various techniques on different samples of psoas muscle Muscle fiber type
Enzyme histochemistry
55 45
I
II A AB B
17 13 15
Immunohistochemistry Frozen sections
Paraffin sections
58 42 -
62 38 -
numbers represent percentage of positive fibers
Discussion
Muscle fiber typing for physiological and pathological studies is conventionally performed by mATPase enzyme histochemistry. When tissue sections are preincubated in p H 4.3, reactive fibers are type I, whereas reactivity after preincubation in p H 10.4 is found in type II fibers. Further subdivision (I, IIA, IIAB, IIB) can be obtained by performing the reaction after preincubation at p H 4.6. Type C (I and II) fibers are intermediate between types I and I I A (Staron and Pette 1986). Elegant enzyme histochemical and biochemical studies on single fibers have allowed Staron and Pette (1986, 1987) to establish the myosin heavy chain phenotypes which correspond with histochemical fiber types. Types I, I I A and IIB fibers contain type I, I I A and IIB M H C respectively. Type IIAB fibers are intermediate in containing types I I A and IIB MHC. Although widely used, enzyme histochemical fiber typing has a number of drawbacks, mainly related to the necessity to use frozen sections. Consequently, an immunohistochemical technique, especially when applicable to paraffin sections, would be of significant practical value. Immunohistochemical approaches to fiber typing have been reported previously. The antibodies used, however, detect an unidentified antigen (Dodson et al. 1987) or a neurofilament protein (Nakamura et al. 1987; Oldfors and Seidal 1989). Clearly, the use of well characterized antibodies against skeletal muscle proteins would be preferable. In the present study we have shown that immunohistochemical muscle fiber typing can be performed on frozen as well a paraffin embedded tissues with the monoclonal antibodies RI ID10 and MY-32. The specificity of R 1 1 D I 0 for cardiac beta M H C and type I M H C is explained by the finding of Jandreski et al. (1987), who demonstrated considerable homology between a cloned cardiac beta M H C gene and a cloned skeletal beta-like M H C gene. The only practical restriction of our immunohistochemical approach is that subtyping of type II fibers in type IIA, IIAB and IIB is impossible with these antibodies. Preliminary observations on electrically stimulated latissimus
dorsi muscle suggest that type IC fibers display weak immunoreactivity with MY-32 and type IIC fibers display weak immunororeactivity with R11D10. With specific antibodies to type I I A or type IIB M H C the problem of subtyping of type II muscle fibers might be further explored. A monoclonal antibody (F75.2.A3) specifically reactive for type IIB fibers in frozen sections has already been described by Pons et al. (1986). Immunohistochemical muscle fiber typing of routinely processed skeletal muscle has several advantages compared to enzymehistochemical typing of frozen tissue. 1) Biopsy freezing, storage of frozen material and cryostat sectioning are no longer necessary. 2) Morphological details are better preserved in paraffin sections than in frozen sections. 3) Immunoelectronmicroscopical studies might be possible. 4) Archival paraffin blocks can be studied which would be, especially in hereditary muscle diseases, very helpful.
Acknowledgment. This study was supported by a grant from the Netherlands Heart Foundation. References
Billeter R, Weber H, Lutz H, Howald H, Eppenberger HM, Jenny E (1980) Myosin types in human skeletal muscle fibers. Histochemistry 65 : 249-259 Brooke MH, Kaiser KK (1970) Muscle fiber types: how many and what kind? Arch Neurol 23:369-379 Dodson A, Garson J, Burke M, Anderton BH (1987) Monoclonal antibody that detects human type I muscle fibers in routinely fixed waxed embedded sections. J Clin Pathol 40:172 174 Gr6schel-Stewart U, Meschede D, Lehr I (1973) Histochemical and immunohistochemical studies on mammalian striated muscle fibers. Histochemie 33 : 79 85 Jandreski MA, Sole MJ, Liew CC (1987) Two different forms of beta myosin heavy chain are expressed in human striated muscle. Human Genet 77:127-131 Khaw BA, Mattis JA, Melincoff G, Strauss HW, Gold HK, Haber E (1984) Monoclonal antibody to cardiac myosin: imaging of experimental myocardial infarction. Hybridoma 3 : 11 23 Moore SE, Orest H, Walsh FS (1984) Immunocytochemical analysis of fibre type differentiation in developing skeletal muscle. J Neuroimmunol 7:13%149 Nakamura T, Kawahara H, Miyashita H, Watarai K, Takagi M, Tachibana S (1987) Cross reactive identification of types 1 and 2 C fibers in human skeletal muscle with monoclonal anti-neurofilament (200 kD) antibody. Histochemistry 87 : 39-45 Oldfors A, Seidal T (1989) Immunohistochemical demonstration of different muscle fibre types in paraffin sections (brief report). Histopathology 15:420423 Pons F, L6ger JOC, Chevallay M, Tom6 FMS, Fardeau M, L6ger JJ (1986) Immunocytochemical analysis of myosin heavy chains in fetal skeletal muscles. J Neurol Sci 76:151 163 Staron RS, Pette D (1986) Correlation between myofibrillar ATPase activity and myosin heavy chain composition in rabbit muscle fibers. Histochemistry 86:1%23 Staron RS, Pette D (1987) The multiplicity of combinations of myosin light chains and heavy chains in histochemically typed single fibers. Biochem J 243 : 695 699
Histochemistry (1990) 93:497-499
© Springer-Verlag 1;990t~
Muscle fiber typing in routinely processed skeletal muscle with monoclonal antibodies M.G. Havenith, R. Visser, J.M.C.
Schrijvers-van Schendel, and F.T. Bosman*
Department of Pathology, University of Limburg, P.O. Box 616, NL-6200 MD Maastricht, The Netherlands Accepted December 2, 1989
Muscle fiber typing is conventionally performed using mATPase enzyme histochemistry on cryostat sections. After pre-incubation of sections at p H 4.3, 4.6 and 10.3, based on the pattern of enzyme reactivity, the fibers can be classified in types I, II (subtypes A, AB and B) and the intermediate C (I and II) fibers. We have attempted to perform fiber typing of human psoas muscle by immunohistochemistry, using monoclonal antibodies R11D10 (specific for cardiac and type I skeletal myosin) and MY-32 (specific for fast muscle fibers) on cryostat as well as on paraffin sections. Staining of consecutive cryostat sections showed that type I fibers are R11D10 reactive whereas type II fibers are MY-32 reactive. Subtyping of type II fibers could not be performed by immunohistochemistry. Quantitative analysis of type I and II fibers showed that enzyme histochemical and immunohistochemical analysis are in close agreement. Summary.
Introduction
Analysis of myofibrillar ATPase (mATPase) activity after preincubation at different p H is the commonly used method for muscle fiber typing (Brooke and Kaiser 1970), However, this technique has to be applied on serial cryostat sections of fresh frozen tissue. The observed differences in mATPase staining intensity between different fibers correlate with the myosin heavy chain (MHC) content, as demonstrated by electrophoretic analysis of single muscle fibers (Staron and Pette 1986). If specific antibodies to M H C were available these could be used for muscle fiber typing by immunohistochemistry. Along these lines, Gr6schel-Stewart et al. (1973) developed antibodies against myosin from human pectoralis muscle, but these antibodies did not allow an unambiguous differentiation between type I and type II fibers. Billeter et al. (1980), however, succeeded in discriminating h u m a n type I and II fibers with polyclonal antibodies to rabbit slow and fast myosin, which had been raised in guinea pigs. Moore et al. (1984) raised murine monoclonal antibodies specific for type I and type II myosin. If the myosin heavy chains remain immunoreactive subsequent to tissue fixation and paraffin embedding, in principle muscle fiber typing can be performed on paraffin sections, which would obviate * To whom offprint requests should be sent
the need for the more complex procedure o f enzyme cytochemistry on frozen sections. Hitherto three reports dealing with muscle fiber typing in paraffin sections have been published. In these studies a monoclonal antibody (F7) against an insoluble fraction of h u m a n brain stem, with an unidentified antigen, and a monoclonal antibody against neurofilament were used (Dodson et al. 1987; N a k a m u r a et al. 1987; Oldfors et al. 1989). In the present report we describe the application o f the monoclonal antimyosin antibodies R l l D I 0 (Khaw et al. 1984) and MY-32 on frozen and paraffin sections for immunohistochemical fiber typing and a comparison with enzyme histochemical muscle fiber typing with the classical mATPase method. Materials and methods
Human psoas muscle specimens were obtained at autopsy (not more than 2 h after death) and frozen in isopentane quenched in liquid nitrogen. Frozen sections of 12 gm thickness were used for enzymehistochemistry and of 4 gm for immunohistochemistry. Thereafter, the remaining frozen tissue was fixed in neutral buffered paraformaldehyde, embedded in paraffin, and sectioned at 4 gm for immunohistochemistry. The mATPase staining reactions were performed at pH 9.4 following preincubation at pH 4.3, 4.6, and 10.4 respectively (Brooke and Kaiser 1970). The mATPase reactivity after preincubation at different pH for the various fiber types, which is related to the MHC phenotype, is listed in Table l. RlID10 (Khaw et al. 1984) was a kind gift from Centocor Europe (Leiden, The Netherlands). For production of the antibody human cardiac myosin was used as immunogen. R l t D I 0 is of the IgG2a subclass and reacts Table 1. Enzyme histochemically determined mATPase activity and
MHC immunoreactivity of human psoas muscle Muscle fiber mATPase typeS pH 4.6 pH 4.3 I IIA
IIAB IIB
RI1D10 MY-32 pH 10.4
+++ --
+++ -+
--
+++
--
+ ++ + ++
-
++ ++
-
+ +
+ + +
-
+ +
Legends: + + + strong reactivity; + + intermediate reactivity; + weak reactivity; - no reactivity. a fiber type assigned according to Staron and Pette (1986, 1987)
498
Fig. 1 A-E. Serial frozen sections of human psoas muscle. A mATPase staining, pH 4.3, B mATPase staining pH, 4.6, C immunoperoxidase staining with RllD10, D mATPase staining pH 10.4, E immunoperoxidase staining with MY-32. × 235
specifically with cardiac myosin and slow (type I) skeletal myosin. MY-32 (Sigma) is a mouse monoclonal antibody of the IgG1 subclass raised against rabbit skeletal muscle. The antibody reacts specifically with fast (type II) myosin. Both monoclonal antibodies show extensive species cross-reactivity. Frozen sections were fixed in acetone at - 2 0 ° C for 20 min. Paraffin sections were deparaffinized and rehydrated. In both, endogenous peroxidase activity was blocked in 0.3 % HzO2 in methanol for 20 min at room temperature (RT). After washing with phosphate buffered saline (PBS) (3 × 5 min) the sections were incubated with the monoclonal antibodies in a moist chamber at RT. To restore the immunoreactivity for R l l D t 0 and MY-32 in paraffin sections, preincubation with 0.1% pepsin (Sigma) in 0.1 N HCI (30 min at RT) was required. R11DI0 was diluted 1:10000 and MY-32 1 : 4000, both in PBS with 1% bovine serum albumin (BSA). After 3 washes in PBS, peroxidase labeled rabbit anti mouse antibodies, diluted 1:200 in PBS with 1% BSA, were applied for 1 h. After final washing, the immunoreactivity was visualized by a diaminobenzidine-H202 substrate with addition of 0.01 M imidazole. In order to compare the overall results of fiber typing by enzyme histochemistry with those of immunohistochemistry on frozen as well as paraffin sections, fiber types of consecutive fibers were visually classified and expressed as a percentage of all fibers.
(I and II) C fibers intermediate between types I and IIA, was almost absent. Therefore the possibility to identify these intermediate fibers by immunohistochemistry was not explored further. On psoas muscle preparations, fiber typing (in types I and II) was performed in parallel sections using enzyme histochemistry on cryostate sections and immunohistochemistry on cryostat and paraffin sections. Reproducible labeling patterns on immunostained sections were obtained with antibodies R11D10 and MY-32 (Fig. 2). The quantitative d a t a on fiber typing, summarized in Table 2, demonstrate that identical results are obtained using these methods.
Results By m A T P a s e enzyme histochemistry on cryostat sections o f h u m a n psoas muscle after preincubation at p H 4.3 and p H 4.6 the usual mosaic pattern was obtained with intensely stained, weakly stained and unstained fibers (Fig. 1 A and 1 B). Intensely staining fibers showed strong reactivity with R l l D 1 0 and no reactivity with MY-32 in parallel frozen sections (Fig. 1 C). These fibers can be classified as type I. Unstained fibers, in contrast, showed intense immunoreactivity with MY-32 (Fig. 1 E). These fibers can be classified as type II. By immunohistochemistry on the basis o f MY-32 reactivity these fibers could not be further subclassified. By enzyme histochemistry subclassification of type II fibers could be performed according to the staining intensity at p H 4.6 and 10.4. In the psoas muscle intermediate m A T P a s e reactivity at p H 4.6, which would identify the
Fig. 2A, B. Serial paraffin sections of human psoas muscle. A immunoperoxidase staining with RllD10, B immunoperoxidase staining with MY-32. × 235
499 Table 2. Quantitative assessment of fiber types using various techniques on different samples of psoas muscle Muscle fiber type
Enzyme histochemistry
55 45
I
II A AB B
17 13 15
Immunohistochemistry Frozen sections
Paraffin sections
58 42 -
62 38 -
numbers represent percentage of positive fibers
Discussion
Muscle fiber typing for physiological and pathological studies is conventionally performed by mATPase enzyme histochemistry. When tissue sections are preincubated in p H 4.3, reactive fibers are type I, whereas reactivity after preincubation in p H 10.4 is found in type II fibers. Further subdivision (I, IIA, IIAB, IIB) can be obtained by performing the reaction after preincubation at p H 4.6. Type C (I and II) fibers are intermediate between types I and I I A (Staron and Pette 1986). Elegant enzyme histochemical and biochemical studies on single fibers have allowed Staron and Pette (1986, 1987) to establish the myosin heavy chain phenotypes which correspond with histochemical fiber types. Types I, I I A and IIB fibers contain type I, I I A and IIB M H C respectively. Type IIAB fibers are intermediate in containing types I I A and IIB MHC. Although widely used, enzyme histochemical fiber typing has a number of drawbacks, mainly related to the necessity to use frozen sections. Consequently, an immunohistochemical technique, especially when applicable to paraffin sections, would be of significant practical value. Immunohistochemical approaches to fiber typing have been reported previously. The antibodies used, however, detect an unidentified antigen (Dodson et al. 1987) or a neurofilament protein (Nakamura et al. 1987; Oldfors and Seidal 1989). Clearly, the use of well characterized antibodies against skeletal muscle proteins would be preferable. In the present study we have shown that immunohistochemical muscle fiber typing can be performed on frozen as well a paraffin embedded tissues with the monoclonal antibodies RI ID10 and MY-32. The specificity of R 1 1 D I 0 for cardiac beta M H C and type I M H C is explained by the finding of Jandreski et al. (1987), who demonstrated considerable homology between a cloned cardiac beta M H C gene and a cloned skeletal beta-like M H C gene. The only practical restriction of our immunohistochemical approach is that subtyping of type II fibers in type IIA, IIAB and IIB is impossible with these antibodies. Preliminary observations on electrically stimulated latissimus
dorsi muscle suggest that type IC fibers display weak immunoreactivity with MY-32 and type IIC fibers display weak immunororeactivity with R11D10. With specific antibodies to type I I A or type IIB M H C the problem of subtyping of type II muscle fibers might be further explored. A monoclonal antibody (F75.2.A3) specifically reactive for type IIB fibers in frozen sections has already been described by Pons et al. (1986). Immunohistochemical muscle fiber typing of routinely processed skeletal muscle has several advantages compared to enzymehistochemical typing of frozen tissue. 1) Biopsy freezing, storage of frozen material and cryostat sectioning are no longer necessary. 2) Morphological details are better preserved in paraffin sections than in frozen sections. 3) Immunoelectronmicroscopical studies might be possible. 4) Archival paraffin blocks can be studied which would be, especially in hereditary muscle diseases, very helpful.
Acknowledgment. This study was supported by a grant from the Netherlands Heart Foundation. References
Billeter R, Weber H, Lutz H, Howald H, Eppenberger HM, Jenny E (1980) Myosin types in human skeletal muscle fibers. Histochemistry 65 : 249-259 Brooke MH, Kaiser KK (1970) Muscle fiber types: how many and what kind? Arch Neurol 23:369-379 Dodson A, Garson J, Burke M, Anderton BH (1987) Monoclonal antibody that detects human type I muscle fibers in routinely fixed waxed embedded sections. J Clin Pathol 40:172 174 Gr6schel-Stewart U, Meschede D, Lehr I (1973) Histochemical and immunohistochemical studies on mammalian striated muscle fibers. Histochemie 33 : 79 85 Jandreski MA, Sole MJ, Liew CC (1987) Two different forms of beta myosin heavy chain are expressed in human striated muscle. Human Genet 77:127-131 Khaw BA, Mattis JA, Melincoff G, Strauss HW, Gold HK, Haber E (1984) Monoclonal antibody to cardiac myosin: imaging of experimental myocardial infarction. Hybridoma 3 : 11 23 Moore SE, Orest H, Walsh FS (1984) Immunocytochemical analysis of fibre type differentiation in developing skeletal muscle. J Neuroimmunol 7:13%149 Nakamura T, Kawahara H, Miyashita H, Watarai K, Takagi M, Tachibana S (1987) Cross reactive identification of types 1 and 2 C fibers in human skeletal muscle with monoclonal anti-neurofilament (200 kD) antibody. Histochemistry 87 : 39-45 Oldfors A, Seidal T (1989) Immunohistochemical demonstration of different muscle fibre types in paraffin sections (brief report). Histopathology 15:420423 Pons F, L6ger JOC, Chevallay M, Tom6 FMS, Fardeau M, L6ger JJ (1986) Immunocytochemical analysis of myosin heavy chains in fetal skeletal muscles. J Neurol Sci 76:151 163 Staron RS, Pette D (1986) Correlation between myofibrillar ATPase activity and myosin heavy chain composition in rabbit muscle fibers. Histochemistry 86:1%23 Staron RS, Pette D (1987) The multiplicity of combinations of myosin light chains and heavy chains in histochemically typed single fibers. Biochem J 243 : 695 699
