Alcian blue staining

In order to confirm that the Caco-2/HT29-MTX co-culture model produced mucus, Alcian blue staining was used. Alcian blue is a blue dye used to stain mucus due to its specificity to acidic glycoproteins [37]. Briefly, the Caco-2/HT29-MTX co-culture was washed twice with PBS and then fixed with 4% formaldehyde for 25 min at RT. The cells were washed thrice with PBS and stained with 10 mg/ml Alcian blue (in 3% acetic acid) for 30 min at RT. The cells were washed with PBS and the inserts were then carefully excised and mounted onto a microscopic slide. The cells were covered with a glass coverslip, visualised with Axiovert 40 C light microscope (ZEISS, Germany) and the image taken with camera (EOS 60D Canon, Japan).