XB-IMG-154647
Xenbase Image ID: 154647
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Fig. 2.
Antibody characterization and controls. (A) Myo6 western blot. Myo6 antibody labels one
band of the expected molecular weight in mouse, Xenopus and zebrafish (~150 kDa). (B)
Myo7a western blot. Anti-myo7a labels a single band of ~200 kDa in zebrafish and American
shad. This band corresponds to the smaller of two labeled bands obtained from mouse cochlea.
(C–H) Epitope expression in cultured cells demonstrating antibody binding to zebrafish
myosins. GFP-labeled cells are positively transfected with the specific construct (myo6a,
myo6a, or myo7a, see below), while GFP-negative cells were not transfected and therefore
serve as negative controls for immunolabeling. (C–E) myo6a, (F) myo6b, (G) myo7a, (H)
control. (C) Myo6a-transfected NIH-3T3 cell, shown here expressing GFP. (D) The myo6a transfected
cell from panel C, immunolabeled for myo6. This demonstrates that the myo6
antibody binds to zebrafish myo6a. (E) Triple-labeled myo6a-transfected cell showing GFP
expression from panel C (green), myo6 immunocytochemistry from panel D (red) and DAPI
labeling (blue), which shows the successfully transfected cell surrounded by non-transfected
cells. (F) Triple-labeled (anti-GFP, anti-myo6, DAPI) NIH3T3 cell expressing myo6b. This
cell demonstrates that the myo6 antibody also binds zebrafish myo6b. (G) Triple-labeled (antiGFP,
anti-myo7a, DAPI) HEK293 cells transfected with myo7a, showing that the myo7a
antibody binds zebrafish myo7a. (H) NIH3T3 cell transfected with myo6b but not labeled with
anti-myo6, showing that GFP fluorescence does not bleed-through to red fluorescent channels.
(I–K) Immunocytochemistry controls in whole-mount inner ear epithelia. Each panel is a
merged image of phalloidin (green) and anti-myo6 (red) images. (I) Zebrafish utricle processed
for myo6 immunocytochemistry but with primary antibody omitted, demonstrating no bleedthrough
of the phalloidin fluorescence to the red channel. (J) American shad utricle labeled with anti-myo6 but with the secondary antibody omitted. The plane of section is at the cuticular
plate level, showing no myo6 staining in this region. (K) American shad utricle with clear
myosin labeling in the cuticular plate region. Phalloidin was omitted, showing that myo6
staining is genuine and not caused by bleed-through from the green channel. Scale bar in C is
25 μm and applies to panels C–H. Scale bars in I–K are 2 μm. Image published in: Coffin AB et al. (2007) Copyright © 2007. This image is reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View |