Pas kit

We analyzed mucin content using PAS assay as described as previously (Garcia et al., 2009 (link)). HT-29 cells were cultured in 6-well plates and treated as mentioned in the western blotting assay. Then treated cells were collected and lysed using RIPA buffer to obtain soluble ingredients. Each sample was placed in the 96-well plate (10 μl/well) followed by added 200 μl 0.1% periodic acid (Solarbio Science & Technology Co., Ltd., Beijing, China). The plate was incubated for 2 h at 37°C, then 200 μl of Schiff’s reagent (Solarbio Science & Technology Co., Ltd., Beijing, China) was added and incubated at room temperature for half an hour. Optical density (OD) of the resulting solution was detected at 555 nm wavelengths and taken as a measure of the amount of PAS positive product. Each assay was performed in triplicate wells and repeated three times.
To explore the morphological alterations of mucin layers during LBS and/or E. coli K1 treatment, HT-29 monolayers were cultured in 24-well plate and treated as described in the western blotting assay. Then monolayers were fixed with 4% paraformaldehyde at 4°C overnight and stained using a PAS kit (Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer’s instruction. The results were observed and analyzed using light microscopy.

MCF-7 cells and MCF-7/ADR cells cultured in a 12 well plate were washed with PBS three times, and intracellular glycogen was measured using a PAS kit (G1360, Solarbio, China) in accordance with the manufacturer’s guidelines (Liu et al., 2018 (link)). PAS staining assay for detecting intracellular glycogen.

Liver tissues were fixed in 4% paraformaldehyde (Solarbio, P1110, China) and were paraffin-embedded. Multiple sections (5 mm) were prepared and stained with hematoxylin and eosin (H&E) for general morphological observation. Liver glycogen was determined using a PAS kit (Solarbio, G1281, China) following the manufacturer’s instructions. Liver tissues were used to detect the liver TC (Nanjing Jiancheng, A111-1-1, China) and liver TG (Nanjing Jiancheng, A110-1-1, China) levels by using the relevant kits.

Indocyanine green (ICG, Sigma) was added to the culture at a final concentration of 1 mg/mL. Cells were incubated at 37 °C for 1 h and then washed three times with PBS. ICG uptake was visualized under a light microscope.
Glycogen storage of HLCs derived from the size-based UC-MSC populations was detected using a periodic acid-Schiff (PAS) kit (Solarbio, China) according to the manufacturer’s instructions. Briefly, cells were fixed in 4% formaldehyde for 30 min, oxidized in 1% periodic acid for 10 min, and rinsed twice with water. Subsequently, cells were treated with Schiff’s reagent for 15 min and then rinsed with water. Glycogen storage was assessed under a light microscope (Olympus, Tokyo, Japan). ImageJ software (National Institutes of Health, MD, USA) was used to quantify the area of positive cells by binarizing images followed by area extraction.

Caco-2 cells grown in 24-well plate were infected with E. coli K1 as described in the adhesion assay. Then monolayers were harvested and fixed in 4% paraformaldehyde at room temperature for 20 m. PAS staining was performed according to the manufacturer’s instructions of PAS kit (Solarbio Science & Technology Co., Ltd, Beijing, China). The PAS-stained wells were counterstained with hematoxylin and observed using light microscopy. For PAS staining of intestinal tissues, 0035 μm sections were fixed in 4% formaldehyde and paraffin-embedded. PAS staining was conducted as described above.