Each panel indicates the day that this sample was collected. a point mutation that leads to a truncated protein.8 In humans, 7 different homozygous recessive mutations in TMIE currently are known to exist in affected members of consanguineous families segregating severe-to-profound prelingual deafness, consistent with linkage to DFNB6.9,10 Although the functions of murine Tmie and human TMIE are unknown, this protein appears to be important for normal hearing and vestibular function. In a previous study, we produced transgenic mice overexpressing that resulted in phenotypic rescue of circling.11 Normal expression of transgenic induced phenotypic rescue in circling homozygous mutants, although some mice did not show amelioration of abnormal behavior, hearing ability, or tissue morphology in the inner ear. Therefore the Tmie protein is required for normal inner ear function in mouse.11 To better understand the function of Tmie, we focused on the spatiotemporal expression of is expressed in various tissues.2,13 Whether Tmie plays an important role in those tissues is uncertain, Berberrubine chloride because circling mice that lack the entire gene have no noteworthy problems in any tissues except those of the inner ear systems.6 In this study, we were interested in the postnatal stages before and after the onset of hearing (around postnatal day [P] 12) in rats; therefore, the postnatal period P0 to19 was studied. Although all the cells that form the mature cochlea are present at birth, important conformational changes occur during this period, including the formation of the tunnel of Corti and the establishment or retraction of neuronal connections. The expression pattern of in the developing inner ear during early postnatal development has not been investigated previously. Here we document our use of a Tmie-specific antibody to elucidate the spatial and temporal expression of in the rat inner ear during postnatal development. Case Report SpragueCDawley rats were used in this study. The animals were provided with a commercial diet and water ad libitum and housed at 22 2 C, relative humidity of 50% 5%, and with a 12:12-h light:dark cycle (lights on, 0730 to 1930). Rats were kept in a specific-pathogenCfree conditioned animal care facility and were free of the following microorganisms: Sendai virus, spp., expression analysis, cochlear samples were collected from P0 to P19. The inner ear tissues of rats were fixed by cardiac perfusion with 2.5% glutaraldehyde and 4% paraformaldehyde in PBS. After 3 to 4 4 d of fixation, the removed temporal bone was fixed in 4% paraformaldehyde for 16 h, decalcified with 10% EDTA in PBS for 2 wk, dehydrated, and embedded in paraffin wax. Sections of 4 m Berberrubine chloride were deparaffinized in xylene and rehydrated through graded concentrations of ethanol. For the immunohistochemical study, LSAB-kit Universal K680 KMT6 (DAKO, Carpinteria, CA) was used according to the manufacturer’s instructions. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 5 min at room temperature. Sections were washed in PBS, and nonspecific binding was blocked with 1% bovine serum albumin for 1 h. Primary antibody (antiTmie, 1:50 dilution) was added to the section and incubated for 1 h. After repeated washes with PBS, the section was incubated with a biotinylated secondary antibody for 1 h and then covered for 15 min with streptavidin peroxidase. Finally, after repeated washes with PBS, the section was stained in a freshly prepared substrate solution (3 mg 3-amino-9-ethylcarbazole in 10 mL 3 M sodium acetate buffer [pH 4.9], 500 L dimethylformamide, 0.03% hydrogen peroxidase) for 10 min. The nuclei of immunostained cells were counterstained with Mayer hematoxylin (Sigma-Aldrich, St Louis, MO). At P0 to 1 1, weak expression was detected in the stereocilia of hair cells in the cochlea (Figure 1 A, B). Stria vascularis, spiral limbus, spiral ligament, and spiral ganglion cells Berberrubine chloride had very weak or no Tmie antibody staining. At P2, expression was also seen in the cell body region of inner and outer hair cells (Figure 1 C). Expression in the stereocilia region was stronger. This pattern continued to P6, and the signal increased in the cell body from P9 to P13 (Figure 1 ECH). At P14, was highly expressed in organ of Corti cells (Figure 1 I). Strong immunoreactivity for Tmie was detected in inner and.