Muscularis Mucosae

Subcutaneous adipose tissue depots were fixed in 4% paraformaldehyde for 24 h at room temperature. Samples were then immersed in ethanol 100% before processing for paraffin embedding. To determine the adipocyte diameter, paraffin sections of 5 µm were stained with hematoxylin and eosin. Images were obtained using a SCN400 slide scanner and digital Image Hub software 561 (Leica Biosystems, Wetzlar, Germany). Adipocyte diameter was determined using ImageJ (National institutes of health, Bethesda, MD, USA). F4/80 positive areas in the adipose tissue were randomly counted after immunostaining with F4/80 antibody (Ab6640, Abcam, Cambridge, UK). All histological observations were analyzed in a blinded manner by three individuals. At least 5 high-magnification fields/mice were randomly selected and obtained using SCN400 slide scanner and digital image hub software (Leica Biosystems, Wetzlar, Germany).
Analysis of the mucus layer and goblet cells was made as previously described.^{89 (link)} Briefly, paraffin sections of 5 μm were stained with alcian blue. Images were captured at ×20 magnification and obtained using a SNC400 slide scanner and digital Image Hub software 561 (Leica Biosystems, Wetzlar, Germany). Analyses were performed using ImageJ (version 1.48 r, National Institutes of Health, Bethesda, Maryland, USA) in a blinded manner. For the mucus layer thickness, two to four fields were used for each mouse and a minimum of 50 different measurements (up to 200) were made perpendicular to the inner mucus layer per field. Each value represents the mean of the different measurements per field. For the goblet cells, the luminal side, muscularis mucosae, submucosa, and muscle layer were removed using ImageJ software and the blue area and the total area were measured separately in the remaining mucosal part of the colon. The proportion of the goblet cells was quantified based on the ratio between the blue area over the total area.

For histological analysis, cross sectional images of the proximal, mid, and distal small intestine and colon were captured by an EVOS^TM 7000 Imaging System at a 10× magnification. Invitrogen Celleste Image Analysis Software (SKU# AMEP4816) was then used for villi and crypt morphometric evaluation. The lines feature was used to measure villi height from the top of villus to the top border of muscularis mucosae, while the crypt depth was measured form the top to the base of the crypt. Eleven to eighteen measurements were taken per mouse. An average for each mouse was then determined. The number of goblet cells was counted in both villi and crypt of distal small intestine and crypt of colon. The stained goblet cells were counted from three individual regions for each mouse and the average cell number per villi or crypt was then calculated.