Quantification of LGR5 expression relative intensity in porcine cochleae
LGR5 expression relative intensity in porcine cochleae from E80, P0, P60, and P120 is collected at six different frequencies of 40 Hz, 100 Hz, 500 Hz, 1000 Hz, 10000 Hz, and 20000 Hz. To quantify the LGR5 expression, the LGR5 intensity of 10 individual cells from each subset of supporting cells including greater epithelial ridge/inner sulcus cells (GER/ISC), inner border cells (IBC), inner phalangeal cells (IPHC), inner and outer pillar cells (IPC, OPC),1st and 2nd Deiters’ cells (DC12), 3rd Deiters’ cells (DC3), and Hensen cells (HS) on surface view was measured via ImageJ and divided by the background intensity at 6 different frequencies. We divided the LGR5 intensity by the background to normalize the signal strength at different depths and positions, under the assumption that the background intensity i.e., autofluorescence should be equal in all cell locations in the organ of Cortis for an individual sample. A total of 16 cochleae (LGR5-H2B-GFP) were subject to quantification: 4 cochleae were taken from E80 pigs, 4 cochleae were taken from P0 pigs, 4 cochleae were taken from two P60 pigs, and 4 from P120 pigs.