Gure 3A). Furthermore, intact stereocilia bundles of OHCs and IHCs had been also clearly observed by FITC-labeled palloidin staining. These information showed that the red GTTR fluorescence was colocalized with FITC alloidin fluorescence, indicating that gentamicin was more preferentially engulfed by cochlear hair cells. Next, other fixed inner ears have been embedded in paraffin for sectioning. The 4-mm-thick sectioned specimens had been stained with DAPI and examined under a fluorescent microscope. As shown in Cholesteryl sulfate (sodium) In Vivo Figure 3Ba, b, GTTR fluorescence intensity of basal turn hair cells was substantially stronger than that in hair cells at theFigure 3 Distribution of gentamicin-conjugated Texas Red (GTTR) in the inner ear soon after in vivo injection. (A) Postnatal day 7 SpragueDawley rats were injected N-Acetylneuraminic acid Purity & Documentation subcutaneously having a single 300 mg kg dose of GTTR (b, c) or Texas Red (TR) answer (a) and after that permitted to recover for 24 h. Then, the temporal bones had been ready and fixed in four paraformaldehyde (PFA) overnight at four 1C. Apical and basal turns of cochlear explants have been prepared and stained with fluorescein isothiocyanate (FITC)-labeled palloidin for 30 min, and specimens have been observed under a fluorescent microscope. (B) The temporal bones were ready from these rats and fixed in 4 PFA overnight at 4 1C. Subsequent, the temporal bones were embedded in paraffin for sectioning at four mm thickness. The sectioned specimens were stained with FITC-labeled phalloidin for 30 min and 40 ,6-diamidino-2-phenylindole (DAPI) for ten min and examined below a fluorescent microscope. Inset shows punctuate GTTR staining observed in the cuticular plate of outer hair cells (OHCs)14 and inner hair cells (IHCs; double arrow), hair cell membrane (arrowhead), outer pillar cells (op), inner pillar cells (ip), Hensen’s cells (h) and the spiral ligament (SL).Experimental Molecular MedicineTRPV channels in gentamicin uptake J-H Lee et alFigure four Gentamicin-conjugated Texas Red (GTTR) accumulation in the inner ear following consecutive in vivo injections. To further test whether GTTR accumulation inside the inner ear is impacted by the amount of injections, postnatal day three Sprague-Dawley rats were injected subcutaneously with GTTR (300 mg kg each day) when (a), twice (b) or 3 times (c) and permitted to recover for 24 h. Inner ears had been fixed in paraformaldehyde (PFA) overnight at four 1C and embedded in paraffin for sectioning at four mm thickness. Specimens have been stained with 40 ,6-diamidino-2-phenylindole (DAPI) and examined beneath a fluorescent microscope. IHCs are indicated by arrowhead and OHCs by arrow. IHCs, inner hair cells; Lim, spiral limbus; OHCs, outer hair cells; SL, spiral ligament; SV, stria vascularis.apical turn. Negligible GTTR fluorescence was observed in numerous on the surrounding supporting cells, spiral ligament, stria vascularis and spiral ganglion neurons (Figure 3B). The P3 SD rats had been injected subcutaneously with GTTR (300 mg kg per day) after, twice or 3 times and permitted to recover for 24 h to additional test whether GTTR accumulation in the inner ear was impacted by the amount of injections. Inner ears had been fixed in PFA overnight at 4 1C and embedded in paraffin for sectioning at 4 mm thickness. The specimens were stained with DAPI and examined below a fluorescent microscope. As shown in Figure four, GTTR accumulation within the inner ear was amplified by rising the number of injections. Interestingly, in contrast to preferential in vitro GTTR uptake by organ of Corti hair cells, in vivo GTTR up.