Validation of a simple, rapid, and economical technique for distinguishing type 1 and 2 fibres in fixed and frozen skeletal muscle

• Published in: Journal of clinical pathology 2002-05, Vol.55 (5), p.375-380
• Main Authors: Behan, W M H, Cossar, D W, Madden, H A, McKay, I C
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - metabolism; Adolescent; Adult; Biological and medical sciences; Biopsy; Biopsy - methods; Cloning; Drinking water; fast and slow myosin; Female; Humans; IHC; Immunohistochemistry; Investigative techniques, diagnostic techniques (general aspects); Laboratories; Male; Medical sciences; Methods; Middle Aged; Monoclonal antibodies; Muscle Fibers, Fast-Twitch - pathology; Muscle Fibers, Slow-Twitch - pathology; Muscle, Skeletal - pathology; Muscular Diseases - pathology; Musculoskeletal system; myofibrillar ATPase; Neuromuscular diseases; normal rabbit serum; NRS; Original; Osteoarticular system. Muscles; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Phosphatase; Physiological aspects; Sensitivity and Specificity; skeletal muscle fibre types; Striated muscle; TBS; Tris buffered saline
• ISSN: 0021-9746; 1472-4146
• DOI: 10.1136/jcp.55.5.375

Aims: To produce a method of distinguishing between type 1 and 2 skeletal muscle fibres that would be more economical and reproducible than the standard ATPase method and be applicable to both fixed and frozen tissue. Because the ATPase method has been accepted as the basis for fibre identification for the past 50 years, the new method should not give significantly different results. Methods: Isoforms of myosin correlate with isoforms of myofibrillar ATPase and an immunohistochemical (IHC) double labelling protocol was devised using monoclonal antibodies to fast and slow myosin. This required one tissue section rather than four. The results of the two methods were compared by means of morphometric analysis of skeletal muscle biopsies from 20 normal healthy volunteers. Results: There were no significant differences (p = 0.57) in the percentages of type 1 (46% using the IHC method v 48% using ATPase) or type 2 fibres (54% v 52%, respectively). The 2a and 2b subtypes were distinguished easily. Analysis of variance revealed that cross sectional area (μm2), diameter (μm), form factor, and density of fibre staining (a measure of substrate—enzyme or protein) were all similar. The method worked equally well on fixed material. Conclusion: An IHC method based on the fast and slow isoforms of myosin shows no significant differences in fibre type analysis from the standard ATPase method although it provides important advantages because it is applicable to fixed (including archival) material, is economical and reproducible, and yields a permanent preparation.