Quantification and distribution of MyBP-C and MyBP-H isoforms in fast-twitch zebrafish myotomal muscle. (A) Quantitative LCMS of 5 dpf larval tails (n = 3) and adult, fast-twitch muscle samples (n = 3) reveal two MyBP protein isoforms present at levels above the threshold for quantification: Mybphb (MyBP-H) and Mybpc2b (fMyBP-C) and presented as the relative molar ratio to total myosin heavy chain. (B) Mosaic fluorescence from immunostained FLAG peptide in 5 dpf larval tail injected with hsp70:mypbhb-3xFLAG construct 24 h after heat shock. Fast-twitch myotomal muscle cells (arrow) have a typical trapezoidal shape and off-axis orientation. (C and D) In fast-twitch larval muscle cells expressing transgenic hsp70:mypbhb-3xFLAG (C) or hsp70:mypbc2b-3xFLAG (D), anti-FLAG antibodies label two bands in each sarcomere, creating a fluorescence doublet pattern along the fiber. (E) For each construct, the aligned and integrated intensity of these two bands (open circles) (see Materials and methods) are well fit with two Gaussian peaks (dashed curves) with separations of 608 ± 13 nm (hsp70i:mypbhb-3xFLAG) and 652 ± 13 nm (hsp70:mypbc2b-3xFLAG). Theoretical intensity profiles generated by an analytical model (solid curves) are fit to the experimental data by assuming that the antibody fluorescence is equally distributed across regions of the thick filament, bounded at points 80 and 530 nm (Mybphb-3XFLAG) or 160 and 490 nm (Mybpc2b-3XFLAG) from the sarcomere center. These regions correspond well to the dimensions and location of the mammalian skeletal muscle C-zone. Data are presented as means ± 1 SD.