A novel triple immunoenzyme staining enables simultaneous identification of all muscle fiber types on a single skeletal muscle cryosection from normal, denervated or reinnervated rats

Abstract

Triple immunofluorescence staining has recently been developed to simultaneously identify all muscle fibers on a single cryosection which is helpful for clinical and basic research, but it has disadvantages such as fast photobleaching and unclear outlines of muscle fibers. Triple immunoenzyme staining (TIE) is likely to avoid these disadvantages. In this study, we aimed to establish a sensitive and specific TIE technique to identify fiber types in normal, denervated, and reinnervated rat muscles, and to develop a systematic sampling method for muscle fiber quantification. Tibialis anterior and soleus from normal, denervated, and reinnervated Lewis rat hind limbs were used. Five consecutive cryosections were cut from each muscle, including one for TIE and four for single immunoenzyme staining (SIE). The TIE was performed using the polymerized reporter enzyme staining system for the first two antigens (A4.74 for MyHC-IIA, BA-F8 for MyHC-I) and alkaline phosphatase staining system for the third antigen (BF-F3 for MyHC-IIB), followed by corresponding detective systems and respective chromogens. The type of muscle fibers was quantified by systematic sampling at 12.5%, 25%, 33% and 50% of all muscle fibers, and was compared with that acquired from counting all the fibers (100%). All muscle fiber phenotypes, including pure and hybrid, could be simultaneously identified on a single TIE cryosection with clear outlines. The fiber types on TIE slides matched well with their respective counterpart on the consecutive SIE slides with a 95% match rate. Systematic sampling of 12.5% fibers could represent the true fiber type distribution of the entire muscle section. Our results suggest that novel TIE can effectively visualize fiber types in normal, denervated or reinnervated rat muscles.

Keywords: immunohistochemistry; muscle fiber phenotyping; myosin heavy chain; nerve regeneration; neural regeneration; rats; triple immunoenzyme staining.

Figure 1

Triple immunoenzyme staining (TIE) and four corresponding single immunoenzyme staining (SIE) results in a normal tibialis anterior muscle. TIE at 20× (a) and 5× magnification (f). SIE with antibody A4.74 (b), BA-F8 (c), BF-F3 (d) and BF-F35 (e) at 20× magnification. I, IIA, IIB, IIX, IIA/X and IIB/X represent muscle fiber type I, IIA, IIB, IIX, IIA/IIX and IIB/IIX, respectively. Fiber phenotyping on TIE (a) matched well with phenotyping on the other four SIEs (b, c, d, e).

Figure 2

Triple immunoenzyme staining (TIE) and four corresponding single immunoenzyme staining (SIE) results in a tibialis anterior muscle subjected to 1 week denervation. TIE at 20× (a) and 5× magnification (f). SIE with antibody A4.74 (b), BA-F8 (c), BF-F3 (d) and BF-F35 (e) at 20× magnification. I, IIA, IIB, IIX, IIA/X and IIB/X represent muscle fiber type I, IIA, IIB, IIX, IIA/IIX and IIB/IIX, respectively. Fiber phenotyping on TIE (a) matched well with phenotyping on the other four SIEs (b, c, d, e).

Figure 3

Triple immunoenzyme staining (TIE) and four corresponding single immunoenzyme staining (SIE) results in a tibialis anterior muscle subjected to 8-week denervation. TIE at 20× (a) and 5× magnification (f). SIE with antibody A4.74 (b), BA-F8 (c), BF-F3 (d₁), BF-F3 + laminin (d₂) and BF-F35 (e) at 20× magnification. I, IIA, IIB, IIX and IIB/X represent muscle fiber type I, IIA, IIB, IIX and IIB/IIX, respectively. Fiber phenotyping on TIE (a) matched well with phenotyping on the other four SIEs (b, c, d, e). Laminin immunostaining (cell membrane counterstaining) was added in d2 to improve the visibility of severely atrophied muscle fibers.

Figure 4

Triple immunoenzyme staining (TIE) and four corresponding single immunoenzyme staining (SIE) of a tibialis anterior muscle that was denervated for 12 weeks. TIE at 20× (a) and 5× magnification (f). SIE with antibody A4.74 (b), BA-F8 (c), BF-F3 (d₁), BF-F3 + hematoxylin (d₂) and BF-F35 (e) at 20× magnification. I, IIA, IIB, IIX and IIB/X represent muscle fiber type I, IIA, IIB, IIX and IIB/IIX, respectively. Fiber phenotyping on TIE (a) matched well with phenotyping on the other four SIEs (b, c, d, e). Hematoxylin staining (nuclear counterstaining) was added in d2 to improve the visibility of severely atrophied muscle fibers.

Figure 5

Triple immunoenzyme staining (TIE) and four corresponding single immunoenzyme staining (SIE) results in a reinnervated tibialis anterior muscle (16 weeks after 4-week delayed nerve repair). TIE at 20 × (a) and 5× magnification (f). SIE with antibody A4.74 (b), BA-F8 (c), BF-F3 (d) and BF-F35 (e) at 20× magnification. I, IIA, IIB, IIX, IIC, IIA/X and IIB/X represent muscle fiber type I, IIA, IIB, IIX, I/IIA, IIA/IIX and IIB/IIX, respectively. Fiber phenotyping on TIE (a) matched well with phenotyping on the other four SIEs (b, c, d, e).