Binding of exogenous HSP27 and αB-crystallin to the I-band region of isolated myofibrils. (A) The experimental design for the stretching of single myofibrils as well as images of stretched myofibrils incubated in relaxing buffer with GFP or with Cy3-conjugated secondary antibodies alone (negative controls). Representative immunofluorescence (FL) and phase-contrast (PC) images of human cardiac (B–D) and rabbit psoas myofibrils (E and F) incubated with recombinant sHSP at the SL indicated on left. Binding was visualized using anti-sHSP primary and Cy3-conjugated secondary antibodies. (B, D, and E) Exogenous HSP27 visualized by anti-HSP27 staining. (C and F) Exogenous αB-crystallin visualized by anti-αB-crystallin staining. In D, the actin filaments were extracted from human cardiac myofibrils using a Ca²⁺-independent gelsolin fragment before incubation with HSP27 and antibodies; images on the right demonstrate the efficient removal of actin, except in a narrow portion of the Z-disk. The profile plot taken along the myofibril axis compared the position of HSP27 in the I-bands (red dashed lines) with that of the remnant actin in the Z-disks after gelsolin treatment (black dashed line). Arrowheads, Z-disk. Bars, 5 µm.