Histological analysis
Tissue harvested from the distal colon (1 cm from the rectum) was cut into segments, fixed with 10% formaldehyde overnight, embedded in paraffin, sectioned at 5 μm, mounted on clean glass slides, and stained with hematoxylin and eosin (H&E) for histological evaluation. Each slide was coded and analyzed in a blinded fashion by two investigators using light microscopy. Each sample was assigned a histological score based on cumulative scores assigned to colon characteristics including degree of epithelial damage, loss of crypt architecture, inflammatory cell infiltration, and edema. For each category, a score of 0 indicated normal appearance, a score of 1 indicated mild change, and a score of 2 indicated severe change, encompassing …show more content…
Homogenized tissue was then plated on blood agar plates and incubated at 37°C overnight in aerobic conditions. Bacterial colonies were counted as colony forming units (CFU) and normalized for varying protein concentrations in the tissue homogenate.
Cytokine level measurement
Liver tissue was homogenized and suspended in PBS containing protease inhibitors (sigma). Supernatants were collected after centrifugation (10,000 rpm for 5 min) and the levels of cytokines such as IL-6 and IL-17A were determined using enzyme-linked immunosorbent assay (ELISA; ebiosciences)21.
Immunofluorescence
The fixed colon sections were deparaffinized with xylene and rehydrated through a series of graded alcohols, and then incubated with FITC-conjugated anti-mouse F4/80 antibody (BD Biosciences, United States) at a 1:100 dilution in PBS for overnight at 4℃, followed by counterstained with DAPI. Sections were imaged and photographed using fluorescent microscope (Leica Microsystems, Heidelberg GmbH, Mannheim, Germany).
Microbiota analysis using bacterial 16s rDNA amplification and miseq250
Tissue harvested from the distal colon (1 cm from the rectum) was cut into segments, fixed with 10% formaldehyde overnight, embedded in paraffin, sectioned at 5 μm, mounted on clean glass slides, and stained with hematoxylin and eosin (H&E) for histological evaluation. Each slide was coded and analyzed in a blinded fashion by two investigators using light microscopy. Each sample was assigned a histological score based on cumulative scores assigned to colon characteristics including degree of epithelial damage, loss of crypt architecture, inflammatory cell infiltration, and edema. For each category, a score of 0 indicated normal appearance, a score of 1 indicated mild change, and a score of 2 indicated severe change, encompassing …show more content…
Homogenized tissue was then plated on blood agar plates and incubated at 37°C overnight in aerobic conditions. Bacterial colonies were counted as colony forming units (CFU) and normalized for varying protein concentrations in the tissue homogenate.
Cytokine level measurement
Liver tissue was homogenized and suspended in PBS containing protease inhibitors (sigma). Supernatants were collected after centrifugation (10,000 rpm for 5 min) and the levels of cytokines such as IL-6 and IL-17A were determined using enzyme-linked immunosorbent assay (ELISA; ebiosciences)21.
Immunofluorescence
The fixed colon sections were deparaffinized with xylene and rehydrated through a series of graded alcohols, and then incubated with FITC-conjugated anti-mouse F4/80 antibody (BD Biosciences, United States) at a 1:100 dilution in PBS for overnight at 4℃, followed by counterstained with DAPI. Sections were imaged and photographed using fluorescent microscope (Leica Microsystems, Heidelberg GmbH, Mannheim, Germany).
Microbiota analysis using bacterial 16s rDNA amplification and miseq250