Image acquisition and analysis

Keratin image analysis protocol

1. All images were acquired using the Zeiss LSM 880 with AiryScan and were 3D z-stacks converted into maximum intensity projections
2. All analysis was performed using Fiji (ImageJ)
3. Select an ROI appropriate for the region of the cell to be analysed (e.g. 80 um²) and duplicate the ROI for separate analysis (Image>Duplicate)
4. Process>Enhance local contrast CLAHE (default settings)
5. Image>Adjust>Threshold (select most appropriate level to distinguish keratin bundles and use the same for all images in the same experiment).
6. Edit>Invert
7. Process>Binary>Make binary

Junction analysis

1. Duplicate the binary image
2. Plugins>Skeleton>Skeletonize
3. Analyze>Skeleton>Analyze skeleton (default settings)
4. From the results window sum up the junctions for each ROI and record in Excel

Dispersion analysis

1. Go back to original binary ROI
2. Analyze>directionality
3. Record dispersion in Excel

Thickness and spacing

1. The original binary ROI can also be used here
2. Plugins>BoneJ>Thickness
3. Record thickness and spacing measurements in Excel

For each keratinocytes, three regions of interest are analysed and averaged per cell, and at least 10 cells are analysed for each independent experiment.  A number of these tools calculate additional parameters, which may be relevant and can be analysed similarly.