Quantitation of row 1 and 2 proteins during C57BL/6 development

Row 2 proteins.zip includes data for CAPZB, TWF2, EPS8L2, and MYO15A-L.

We estimated the total immunofluorescence signal in the top 1-2 µm of each tip. Airyscan z-stacks were imported into Fiji, which was used for all analysis steps. For analysis of P0.5 and P7.5 C57BL/6J mice, average Z-projections of Airyscan stacks were made that included row 1 and row 2 tips in the same projection. For analysis of P15.5 and P21.5 C57BL/6J mice, separate Z-projections were made for row 1 and row 2 tips, using the same number of projected x-y slices per row. Regions of interest (ROIs) used circles that encompassed most of each tip. Area and average intensity measurements were made from ten or more row 1 tips and ten or more row 2 tips, and blank measurements were taken from volumes outside the stereocilia and above the epithelium. The total signal (area times average intensity) in each stereocilia tip volume was calculated after subtracting the background. For each hair bundle, the average signal in rows 1 and 2 was calculated, which was used to normalize the individual row 1 and row 2 stereocilia tip signals within each bundle. The average signal row 1 and 2 signal for each bundle was also used for comparison of expression levels at different developmental times. For developmental comparisons, the average signal in rows 1 and 2 for each bundle was normalized to the highest average value for the protein’s developmental series.