Colons were collected from sacrificed mice at the end of each treatment schedule, as described above, fixed in 10% neutral-buffered formalin, and embedded in paraffin. Serial 5 µm microtome-cut sections were then stained with hematoxylin and eosin (Sigma-Aldrich). Slides were acquired with an Aperio AT2 digital scanner (Leica Biosystems) and analyzed with an Aperio Imagescope (Leica Biosystem). Scoring of colonic inflammation was determined as reported by Nagahama et al.44 (link) with minor modifications, by assessing mucosa thickening, inflammatory cells, and submucosa cell infiltration. Each criterion was scored as 0–4, and the sum of each score was defined as the histological score.
Immunohistochemical staining of HIF-1α was performed on colon sections of mice with the anti-HIF-1 alpha antibody (Abcam ab2185, 1:2000) and antigen unmasking in citrate buffer. Sections were incubated with specific Detection KIT-HRP (Abcam, ab236469) according to the manufacturer’s instructions, then counter-stained with hematoxylin to visualize nuclei, dehydrated and mounted in DPX (Sigma-Aldrich). Bright-field images were acquired with an Aperio AT2 digital scanner (Leica Biosystems) and analyzed with an Imagescope (Leica Biosystem) or using a Zeiss Axio Imager M2m microscope with a ×10 or ×20 objective.
Immunohistochemical staining of HIF-1α was performed on colon sections of mice with the anti-HIF-1 alpha antibody (Abcam ab2185, 1:2000) and antigen unmasking in citrate buffer. Sections were incubated with specific Detection KIT-HRP (Abcam, ab236469) according to the manufacturer’s instructions, then counter-stained with hematoxylin to visualize nuclei, dehydrated and mounted in DPX (Sigma-Aldrich). Bright-field images were acquired with an Aperio AT2 digital scanner (Leica Biosystems) and analyzed with an Imagescope (Leica Biosystem) or using a Zeiss Axio Imager M2m microscope with a ×10 or ×20 objective.
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