Total fluorescence intensity (TFI) evaluation
Images of ZO-1, occludin, claudin-3 (Cldn3), MUC5AC and βIV-tubulin on paraffin sections were captured at 400 × magnification with a fluorescence microscope (Olympus IX51, Tokyo, Japan). Protein expression of these markers was analyzed using ImageJ software by calculating the value of positively stained area and the mean fluorescence intensity for each marker. TFI measurements were performed by multiplying the positive area by mean fluorescence intensity and corrected by subtracting the background autofluorescence.