Immunohistochemical myofiber typing and high-resolution myofibrillar lesion detection in LR White embedded muscle

We have developed a method of fixing, embedding, sectioning, and staining t hat allows high-resolution detection of myofibrillar structure and myosin i mmunocytochemical muscle fiber typing in serial semithin sections of LR Whi te plastic embedded muscle at the light microscopic level. Traditional appr oaches, such as cryostat sections, permit fiber typing, but small myofibril lar lesions (1-3 sarcomeres) are difficult to detect because of section thi ckness. Semithin sections of hydrophobic resins do not stain well either hi stochemically or immunocytochemically. Electron microscopy can resolve lesi ons and discriminate fiber types based on morphology, but the sampling area is small. Our goal was to develop a rapid method for defining both fiber t ype and high-resolution primary myofibrillar lesion damage. Mild fixation ( 1-4% paraformaldehyde, 0.05-0.1% glutaraldehyde) and embedment in a hydroph ilic resin (LR White) were used. Myofibrillar structure was extremely well preserved at the light microscopic (LM) level, and lesions could be readily resolved in Toluidine blue stained 500-nm sections. Fiber type was defined by LM immunomyosin staining of serial plastic semithin sections, which dem onstrated reciprocal staining patterns for "fast" (Sigma M4276) and "total" (skeletal muscle) myosins (Sigma M7523). (C) 2000 Wiley-Liss, Inc.