Validation of Primary Antibodies for Multiple Immunofluorescent Labeling of Horse Skeletal Muscle Fiber Type

Immunohistochemical analysis offers the ability to evaluate muscle fiber type, fiber type-specific cross sectional area, organization of fibers, and fiber hybridization. Currently, few primary antibodies have been validated for fiber type analysis in horse skeletal muscle, and those that are available can be cost-prohibitive and laborious to perform on the same sample. The objective of this experiment was to validate a more efficient method of immunofluorescent fiber type staining in horse skeletal muscle. Samples from the gluteus medius and triceps brachii from a mature mare and her fetus in late gestation were embedded in OCT, frozen in liquid nitrogen-cooled isopentane, and stored at -80^º C. Seven-micrometer cross sections were cut in a cryostat, allowed to dry at room temperature for 1 h, and stored at -20^ºC until analysis. One of two protocols was then used to determine fiber type. The first protocol utilized a previously-validated method of immunofluorescent fiber type analysis in horse skeletal muscle (Tulloch et al., 2011), while the second protocol utilized isoform-specific primary antibodies that are commonly used in human and rodent skeletal muscle samples. Protocol 1 utilized primary antibodies against slow (MAB1628; Millpore, Darmstadt, Germany) and fast muscle fibers (MHCf; Leica Biosystems, Wetzlar, Germany). Protocol 2 utilized primary antibodies against myosin heavy chain type I (MyHC1; BA-D5), MyHC2a (SC-71), and MyHC2x (6H1), all from Developmental Studies Hybridoma Bank (Iowa City, Iowa). Following incubation in primary antibodies, sections were incubated in fluorescent secondary antibodies, mounted with fluorescent mounting media, and imaged using a fluorescent microscope (Nikon Instruments, Melville, NY, USA). At least 50 muscle fibers were compared for each sample. Myosin heavy chain type 1 BA-D5 and slow MAB1628 identified the same fibers for all fibers counted. To confirm that the 6H1 antibody labeled type 2x fibers and not type 2a fibers, sections were incubated in a previously validated antibody for type 2a fibers (SC-71; DSHB), as well as the proposed primary antibodies (BA.D5 and 6H1; DSHB). Together, 6H1 and SC-71 labeled the same fibers as MHCf, confirming 6H1 and SC-71 label all fast-twitch fibers. Therefore, the primary antibodies BA.D5 and 6H1, in combination with SC-71, allow for immunofluorescent labeling of multiple fiber types on a single section, using isoform-specific secondary fluorescent labels.

Tulloch, L., J. Perkins, and R. Piercy. 2011. Multiple immunofluorescence labelling enables simultaneous identification of all mature fibre types in a single equine skeletal muscle cryosection. Equine veterinary journal 43: 500-503.