Immunohistochemistry
The positive IF staining for cytokeratin confirms the epithelial origin of the cells (figure 2a). In general, protamine treatment increases extracellular margins between adjacent cells and adherence junction formation is more disrupted (figure 2d-f). IF analyses showed a clear signal of occludin all along the cell borders in the controls (figure 2g). In both intervention groups this signal was reduced, and tight junctions were more interrupted across the cell borders, but still clearly visible. CS56 shows chondroitin sulfate is present in controls, but decreases after protamine treatment (figure 2j-k). Even after removing CS treatment by rinsing the inserts, IF analyses demonstrated an increased signal in the CS treated samples compared to controls and protamine alone samples (figure 2l).