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

Abstract

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 (µm 2 ), 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.

Inorganic Nitrate Mimics Exercise-Stimulated Muscular Fiber-Type Switching and Myokine and γ-Aminobutyric Acid Release

“…Soleus and gastrocnemius cross sections from nitrate treated rats were prepared at a thickness of 10 m using a cryostat. Immunohistochemical staining and fiber-type assessment was performed according to published methods (24). Antibodies were type I fiber, monoclonal antimyosin (skeletal, slow; clone NOQ7.5.4D; Sigma-Aldrich) and type II fiber, monoclonal antimyosin (skeletal, fast; alkaline phosphatase conjugate; clone MY-32; Sigma-Aldrich).…”

Quantifiable diagnosis of neuromuscular diseases through network analysis

“…To the best of our knowledge, this is the largest and most comprehensive dataset of images analyzed in a neuromuscular study. Previous reports have been published of attempts to facilitate the automated extraction of geometric characteristics from muscle biopsies [11][12][13][14][15][16]. These studies rely on the development of segmentation methods using a very small number of samples to only extract morphometric information.…”

“…A large panel of different histochemical and histoenzymatic techniques are necessary to identify pathologic changes in the routine diagnostic process [9,10] (Additional file 1: Figure S1). Previous attempts to automate the extraction of geometrical characteristics from normal muscle biopsies have been published [11][12][13][14][15][16], but those methods fail to provide an automated analysis or adequate scrutiny of the information derived from the analysis. Our analysis begins at this point, taking into account a large number of samples to study both geometrical and network data to include morphometric and organizational information.…”

Assessment of Nerve Regeneration through a Novel Microchannel Array

“…It was discovered that fibrous nerve encapsulation was thicker within the 200 μm regenerating microchannels presented here than the previously published work on ~100 μm channels [9], possibly indicating a geometry-independent process for chronic inflammatory changes of a foreign body response or mechanical pressure response (Figure 5i), which built up over the 5 month regeneration period. To confirm regeneration in the GN, medial muscle fibers were evaluated enzymatically for activity using an ATPase stain to identify Type 1 and Type 2 muscle fibers [28]; fibers exhibited the characteristic loss of "checkerboard" pattern of Type 1 and Type 2 muscle fibers, as expected with nerve transection and regeneration (Figure 5j) [29].…”