Spinning disk confocal unit

Fixed cells transfected with WT or mutant HA-SLC26A3 were blocked for 45 min with 7% normal horse serum, then incubated overnight at 4°C with monoclonal anti-HA (1:300, Cell Signaling Technologies), washed three times in PBS, then incubated 1 h at room temperature with Alexa 546-coupled goat anti-rabbit Ig (Life Technologies). After three final washes, coverslips were inverted and mounted on glass slides using VECTASHIELD mounting medium (Vector Labs, Burlingame, CA). Other antigens were detected with compatible, species-specific fluorophor-coupled secondary Ig. After fixation and immunostaining of filter-grown cells as described above, the filters were excised from their frames and mounted on glass slides under coverslips with VECTASHIELD. Cells were imaged with the Zeiss LSM510 confocal fluorescence microscope or with the Nikon TE2000 inverted microscope interfaced to a PerkinElmer spinning disk confocal unit and an Orca charge-coupled device camera (Hamamatsu, Bridgewater, NJ). Image processing was with Zeiss software or (for images captured by Nikon-PerkinElmer microscope) with Slidebook (Intelligent Imaging Innovations, Denver, CO).

Data from immunohistochemistry experiments were collected on an Olympus (Center Valley, PA, USA) IX81 inverted microscope equipped with an Olympus spinning disk confocal unit, Hamamatsu EM-CCD digital camera (Bridgewater, NJ, USA), and high precision BioPrecision2 XYZ motorized stage with linear XYZ encoders (Ludl Electronic Products Ltd., Hawthorne, NJ, USA) using a 60× 1.40 N.A. SC oil immersion objective. The equipment was controlled by SlideBook 6.0 (Intelligent Imaging Innovations, Inc., Denver, CO, USA), which was the same software used for post-image processing. 3D image stacks (2D images successively captured at intervals separated by 0.25 μm in the z-dimension) that were 512 × 512 pixels (~137 × 137 μm; pixel size = 0.267 μm) were acquired over 50 percent of the total thickness of the tissue section starting at the coverslip. Importantly, imaging the same percentage, rather than the same number of microns, of the tissue section thickness controls for the potential confound of storage and/or mounting related volume differences (i.e., z-axis shrinkage). The stacks were collected using optimal exposure settings (i.e., those that yielded the greatest dynamic range with no saturated pixels), with differences in exposures normalized during image processing.