Quantifying Tenocyte Progenitor Gene Expression

Embryos processed for fluorescent immunohistochemistry were imaged using a Nikon A1 confocal system (RRID: SCR_020318) with a Nikon Eclipse Ti inverted microscope using a CFI Plan Apochromat VC 60XC (water immersion) objective. Embryos processed for isHCR were imaged using Leica TCS SP8 confocal system (RRID: SCR_018169) with a Leica DMi8 inverted microscope using a Plan Apochromat HC 40× (water immersion) objective. Confocal stacks were collected with system optimized spacing of 0.45 microns and were analyzed using ImageJ (RRID: SCR_003070) and Imaris 10 (RRID: SCR_007370) softwares. Tenocyte progenitors at prospective entheses were identified by their proximity to cartilage chondrocytes. To quantify expression of either scxa or sox9a in the selected cell, using ImageJ a projection of a z-stack comprising the entire cell was created (∼8-10 slices) using DAPI signal to identify the edges of the cell. Quantification of fluorescence in the selected cells was performed by creating ROIs around the DAPI signal corresponding to the tenocyte as previously described (Subramanian et al., 2018 (link)). In experiments where Imaris 10 was used, the ROI was created using DAPI as a reference for each tenocyte at each z-section. These ROI surfaces were combined and the mean intensity of scxa, sox9a and col1a was measured in these cellular volumes.