The entire length of the cochlear whole-mount preparation was examined using an epifluorescence illumination microscope (Z6 APO apochromatic zoom system, Leica Microsystems, Buffalo Grove, IL, USA) equipped with a Leica digital camera (DFC3000 G microscope camera) and controlled by Leica Application Suite V4 PC-based software. To visualize detailed structures, the tissues were further examined and photographed using a confocal microscope (LSM510 multichannel laser scanning confocal image system, Zeiss, Thornwood, NY, USA). At each site of interest, a series of confocal images covering the entire thickness of the tissues was collected.
The collected images were processed using ZEN Blue 2012 image processing software (Zeiss, Thornwood, NY, USA) utilizing a previously reported methodology (Yang et al., 2015 (link)). Some collected images were further processed to improve the contrast and clarity of cells using the “Levels Adjustment” and “Despeckle” functions offered in Adobe Photoshop CS6 (version 13.0.1, Adobe Systems, San Jose, CA, USA) to correct the tonal range and reduce image noise. These image-processing steps did not create any analytical bias, because the focus of this study was to examine macrophage morphologies, not expression levels of macrophage marker proteins.
The collected images were processed using ZEN Blue 2012 image processing software (Zeiss, Thornwood, NY, USA) utilizing a previously reported methodology (Yang et al., 2015 (link)). Some collected images were further processed to improve the contrast and clarity of cells using the “Levels Adjustment” and “Despeckle” functions offered in Adobe Photoshop CS6 (version 13.0.1, Adobe Systems, San Jose, CA, USA) to correct the tonal range and reduce image noise. These image-processing steps did not create any analytical bias, because the focus of this study was to examine macrophage morphologies, not expression levels of macrophage marker proteins.