Multiple immunofluorescence labelling enables simultaneous identification of all mature fibre types in a single equine skeletal muscle cryosection

Abstract

Skeletal muscle is composed of a heterogeneous mixture of several fibre types, each with specific physiological properties. In equine muscle, identification of these individual fibres (fibre typing) is important for both exercise physiology and pathological studies. Traditionally, fibre typing has been achieved by adenosine triphosphatase (ATPase) histochemistry or by immunoperoxidase labelling with antibodies directed at myosin heavy chain isoforms. ATPase histochemistry can be temperamental and lacks specificity, and both techniques require staining of serial cryosections to reveal the entire fibre type compliment of a single sample, which is time consuming and prone to inaccuracy. Here we describe an immunofluorescence labelling technique that enables rapid, accurate and specific identification of the 3 mature equine muscle fibre types in a single cryosection.

Functional electrical stimulation following nerve injury in a large animal model

“…Thereafter, 7 μm cryosections were air‐dried onto glass slides and stored at −80°C and processed as previously described . All mature skeletal muscle fiber types (I, IIa and IIx), hybrid fibers and collagen V were identified in single cryosections using a multiple immunofluorescence labelling technique with 4 different primary antibodies . Fibers (n > 400) were manually measured, to obtain the minimal fiber diameter (MFD) and assigned a fiber type or hybrid fiber designation by relative fluorescence.…”

Contractile properties of slow and fast skeletal muscles from protease activated receptor‐1 null mice

“…Cryostat sections of EDL and soleus muscles from 12‐ to 14‐week‐old PAR‐1 null ( n = 6) and control ( n = 4) mice were prepared as described above and then fixed in ice‐cold 50% acetone in methanol (v/v) for 10 min. Sections were then washed in PBS and blocked in 5% normal rabbit serum in PBS (v/v) before immunostaining with antibodies raised against laminin and 3 different myosin heavy chain isoforms using the method of Tulloch et al Briefly, mouse IgG monoclonal antibodies raised against the slow/type I (NOQ7.5.4D; Abcam, Cambridge, UK), fast/type IIa (SC‐71; Developmental Studies Hybridoma Bank, University of Iowa), and “pan” fast (type IIa/b/x) (MY‐32; Abcam, Cambridge, UK) myosin heavy chain isoforms were labeled with anti‐mouse IgG Alexa Fluor 350, 594, or 488 conjugated labeled Fab fragments respectively (Zenon, Life Technologies, Carlsbad, California), according to the manufacturer's instructions. The final concentration of each of the MyHC antibodies was 50 μg/ml.…”

Histopathological assessment of intrinsic laryngeal musculature in horses with dynamic laryngeal collapse

“…Multiple immunofluorescence labelling of myosin heavy chain isoforms was performed according to modifications of a previously described protocol, allowing the specific identification of the 3 adult muscle fibre types in a single cryosection [19]. Multiple immunofluorescence labelling of myosin heavy chain isoforms was performed according to modifications of a previously described protocol, allowing the specific identification of the 3 adult muscle fibre types in a single cryosection [19].…”