Figure 5
Expression and localization of transient receptor potential vanilloid 1(TRPV1) and TRPV4 in inner ear hair cells. (a) Total RNA was isolated from each turn of the cochlea, and complementary DNA (cDNA) was synthesized by reverse transcriptase-PCR (RT-PCR). The TRPV1 and TRPV4 genes were amplified with specific primer sets. GAPDH was used for coamplification of gene transcripts. (b) The stereocilia and bodies of hair cells were stained with anti-TRPV1 antibody14 or anti-TRPV4 antibody (arrowhead indicates outer hair cells (OHCs) and large arrow indicates inner hair cells (IHCs)) overnight at 4 °C. Specimens were washed three times with Tris-buffered saline (TBS) plus 0.05% Tween-20 (TBS-T) and incubated with secondary antibodies for 1 h at room temperature in the dark. Alexa Fluor 488-conjugated donkey anti-goat and Alexa Fluor 568-conjugated goat anti-rabbit were used as the secondary antibodies, respectively. (c) Horizontal tissue sections showing TRPV1 and TRPV4 immunofluorescence staining. Inner ears derived from postnatal day 3 Sprague-Dawley rats were fixed in paraformaldehyde (PFA) overnight at 4 °C and embedded in paraffin for sectioning at 4 μm thickness. The specimens were stained with anti-TRPV1 or anti-TRPV4 antibodies and further stained with 4′,6-diamidino-2-phenylindole (DAPI). These specimens were examined under a fluorescent microscope. O1, first layer of outer hair cells; O2, second layer of outer hair cells; O3, third layer of outer hair cells.
