Indirect immunohistochemical analysis was performed as previously described.
30 Three mAbs against embryonic MyHC were used in this study. mAb 2B6 has been characterized to react specifically with embryonic MyHC.
31 In addition, two mAbs against embryonic MyHC were characterized in this study: mAb 3D1 was raised in our laboratory against cat masseter myosin and was produced according to Lucas et al.
29 and mAb NCL-MHCd (Novocastra Laboratories, Ltd., Newcastle-upon-Tyne, UK) against developmental MyHCs,
32 (abbreviated as NCL-dev in this study). Both mAbs 3D1 and NCL-dev were characterized by Western blot on high-resolution SDS-gels to react with the embryonic MyHC band from the 30-dpc rabbit limb
(Figs. 1 2) . One mAb against neonatal MyHC, NCL-MHCn (Novocastra Laboratories, Ltd.),
33 was used (abbreviated as NCL-neo in this study). Two mAbs against EO-specific fast MyHC were used: 4A6
2 and 10A10, which was raised in our laboratory against rabbit EOM MyHC, according to Lucas et al.
29 and has identical specificity on Western blots and muscle sections as 4A6 (data not shown). mAb BA-G5 was raised against cow atrium and reacts with cardiac specific α-MyHC in rats,
34 obtainable from the American Type Culture Collection (Manassas, VA). In addition we used an mAb against acetylcholinesterase (AChE) to label nerve endings (NCL-AchE; Novocastra Laboratories, Ltd.). Horse-radish peroxidase (HRP)–labeled rabbit anti-mouse immunoglobulin antibody (Dako Corp., Carpinteria, CA) was used as a secondary antibody.