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.
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.
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