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. Author manuscript; available in PMC: 2014 Jul 21.
Published in final edited form as: J Comp Neurol. 2011 Apr 15;519(6):1095–1114. doi: 10.1002/cne.22554

Figure 2.

Figure 2

TRPML3 protein localizes to stria vascularis and hair cell vesicles of neonatal cochleae. Immunohistochemistry on sections of mouse inner ear with three different antibodies raised against different parts of TRPML3 (schematically shown in M). A-E: The carboxyl-terminal CT1 labels inner and outer hair cells (IHC and OHC) as well as cells of the stria vascularis (SV) in Trpml3+/+ (A,C,E) but not Trpml3−/− (B,D) cochleae. White perimeters indicate the position of hair cell nuclei. White dashed lines delineate the apical edge of marginal cells in the stria vascularis. E: DAPI (in white) labels the nuclei of SV cells. F-J: The amino-terminal NT also labels IHC, OHC, and SV cells in addition to Pillar cells (PC) in Trpml3+/+ (F,H,J) but labels only PCs in Trpml3−/− (G,I) cochleae, indicating that the PC immunore-activity is nonspecific. J: DAPI (in white) labels the nuclei of SV cells. K,L: The carboxyl-terminal CT2 also labels IHC, OHC, and PC Trpml3+/+ (K) but labels only PCs in Trpml3−/− (L) cochleae, indicating that the PC immunoreactivity is nonspecific. M: Schematic representation of the TRPML3 protein (membrane-spanning domains depicted as cylinders) and of the antibodies raised against it (represented with Y signs and termed NT, EX, CT1, and CT2). The N and C termini are predicted to be cytoplasmic. N,O: Double immunohistochemistry with antibodies to NT (N) and the late endosome and lysosome marker LAMP1 (O) reveals colocalization of both proteins in vesicles of three outer hair cells. Approximately 66% of TRPML3-positive vesicles were also LAMP1 positive. Arrowheads point to vesicles that contain TRPML3 and LAMP1, whereas arrows point to vesicles that contain LAMP1 but not TRPML3. P-S: Double immunohistochemistry with antibodies to NT (P,R) and LAMP1 (Q,S) also reveals colocalization of both proteins in vesicles of hair cells from utricle (UTR) and crista ampullaris (CA). Arrowheads point to TRPML3-positive vesicles that are large enough to be visible as rings. All pairs of Trpml3+/+ and Trpml3−/− images were acquired with the same exposure, laser, and gain settings. The brightness and contrast settings were adjusted identically for each pair of images. A-L and P-S are from postnatal day 4 (P4) cochleae. N,O are from P7 cochleae. A magenta-green version of this figure is supplied as Supporting Information Figure 1. Scale bars = 50 μm in A,B,F,G; 10 μm in C-E,H–L; 2 μm in N-S.