Expand upon that observation, we developed in rabbits an affinity-purified polyclonal antibody (rafMI ) that was specific for the tail of frog myosinI . Unfortunately, rafMI did not Linuron Protocol cross-react with guinea pig myosin-I , which restricted its use to frog tissue. In all tissues examined, like the saccule, rafMI recognized a frog antigen of 105 kD (Fig. 1 E), which comigrated with purified frog myosin-I (Gillespie, P.G., unpublished results). Like purified frog myosin-I , the antigen recognized by rafMI shifted in migration from 120 to 105 kD upon switching from high to low acrylamide cross-linker concentration (not shown), a characteristic of this isozyme (Gillespie, P.G., unpublished results). The rafMI antibody also detected a single immunoreactive band of 105 kD in purified hair bundles (Fig. 1 A), confirming earlier observations (Gillespie et al., 1993). Quantitative immunoblotting with rafMI indicated that myosin-I was present at three pg per saccular equivalent of hair bundles (data not shown). To figure out the distribution of myosin-I inside sensory epithelia, we made use of indirect immunofluorescence with rafMI (Fig. 2). In agreement with prior studies, we observed myosin-I in stereocilia and hair cell bodies. The highest hair cell concentration of myosin-I was located amongst actin with the cuticular plate and circumferential actin belt, within a domain we term the pericuticular necklace. We also observed labeling at apical surfaces of peripheral cells, that are undifferentiated epithelial cells outdoors the sensory epithelium. These distinct labeling patterns have been absent in nonimmune controls or when fusion protein was included in excess within the labeling reaction. Distribution of myosin-I inside every single of those domains is viewed as separately beneath. Stereocilia. Myosin-I was found mostly in the distal third of each stereocilium and was most concentrated in the bundle’s beveled edge, exactly where punctate label apparently represented the tips of person stereocilia (Fig. two, H, I, and K). In most cells, immunoreactivity in stereocilia was somewhat low compared to that with the cell physique; in smaller sized hair cells with modest bundles at the edge from the sensory epithelium (not shown) or inside the sensory epithelium (Fig. 2, B, C, and H, asterisks), nonetheless, the entire bundle contained high concentrations of myosin-I , con-Electron MicroscopyBullfrog sacculi have been dissected, fixed, and labeled with primary antibodies as described above for Vibratome sections. For labeling of stereocilia, exactly where deep penetration of antibodies into tissue was not essential, the secondary label was protein A conjugated to 5-nm gold particles (J. Slot, University of Utrecht, The Netherlands). The tissue was postfixed with two osmium tetroxide (OsO4) in 1.5 potassium ferrocyanide for 1 h at area temperature, rinsed with 100 mM cacodylate buffer, and then stained enbloc with 2 uranyl acetate in maleate buffer (pH 6.0) for 2 h at four C. Just after dehydration in an ethanol series, the tissue was rinsed briefly in one hundred propylene oxide and flat embedded in an Eponaraldite mixture (EMbed812; Electron Microscope Sciences, Fort Washington, PA) and cured for 48 h at 60 C. Thin sections (silver-gold) were collected onto 200-mesh copper grids from the center on the sensory epithelium along the axis operating parallel towards the eighth-nerve fibers. The sections were poststained with 2 uranyl acetate and lead citrate and viewed with a 100CX electron microscope (JEOL USA, Alprenolol Biological Activity Peabody, MA). In cases requ.