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. 2008 Sep 11;9(4):436–451. doi: 10.1007/s10162-008-0137-8

FIG. 2.

FIG. 2.

Localization of zebrafish Sparc mRNA in the ear and protein in otoliths. a The mRNAs of sparc and β-actin detected in whole embryos during early development by RT-PCR. b–d The mRNA of sparc detected in the developing ear by in situ hybridization. Dorsal views of hindbrain area, anterior at the top; outline in b, left otic placode; arrowheads in d, posterior maculae along the medial wall of the otic vesicle; arrows in d, semicircular canals; scale bar in b for all three panels, 50 μm. e Sparc protein detected in an adult-otolith extract by immunoblotting. About 0.6 μg total protein per membrane strip; Silver, silver-stained gel slice; Sparc, anti-Sparc serum UI-37; E. coli-adsorbed, UI-37 pre-adsorbed with a plain bacterial extract; Sparc-adsorbed, UI-37 pre-adsorbed with a bacterial extract containing recombinant zebrafish Sparc; dashes on the right, molecular-mass markers (kDa, from top) 250, 160, 105, 75, 50, 35, 30, 25, 15, 10. f Sparc protein detected in otic vesicles by whole-mount immunofluorescence. Green, Sparc labeled with antiserum UI-37; blue, nuclei labeled with DAPI; arrowheads, lapillus (left) and sagitta (right); lateral view of otic vesicle, dorsal at the top, anterior to the left; projections of 10-μm stacks of optical sections; scale bar for panels f and g, 25 μm. g Same as f, but with purified naive rabbit IgG instead of UI-37. h–i Sparc protein detected in a transverse section of an adult lapillus. h brightfield view of an otolith slice; i immunofluorescence with antiserum UI-37 at the surface of the slice’s cross-section; scale bar in h for panels h to m, 100 μm. (j–k) Same as h–i, but UI-37 pre-adsorbed with a plain bacterial extract. l–m Same as h–i, but UI-37 pre-adsorbed with a bacterial extract containing recombinant zebrafish Sparc. Because the brightfield images in h, j, and l were overexposed to visualize the growth rings, the visible otolith margins do not exactly match the fluorescent areas; i, k, and m are projections of 58 to 87-μm-thick stacks of optical sections that were acquired under matched settings.