Histology: PAS/Alcian Blue Stain

Sections of distal colon from at least five animals of the different experimental groups were fixed and embedded in paraffin to obtain transversal slices of 5 μm thickness. Then, they were stained with Alcian Blue (AB)/periodic acid-Schiff (PAS), using standard histological techniques, for evaluating mucin expression in goblet cells. Acidic mucins stain blue with AB (pH 2.5) and neutral mucins stain pink with PAS, while mixtures of neutral and acidic mucins appear purple.

The sections from all experimental groups were stained in one single batch to ensure that differences in the staining pattern were not due to technical manipulations, thereby making certain the comparability of the different samples. Slides were examined under an optical microscope (Nikon Eclipse 90i, Nikon Instruments, Inc., Amstelveen, The Netherlands). For quantitative analysis, images were acquired from at least three random fields of view per slice and processed using the ImageJ image analysis program (NIH, Bethesda, MD, USA). The area AB/PAS positive was identified as the ratio to the total tissue area.