Axiocam 506 clolor

After the macroscopic damage evaluation, segments of the distal colon were stapled flat, mucosal side up, onto cardboard and fixed in 10% neutral-buffered formalin for 24 h at 4 °C. Then, samples were dehydrated in sucrose, embedded in paraffin, sectioned at 5 μm, and mounted onto slides. Subsequently, sections were stained with hematoxylin and eosin and examined using an Axio Imager A2 microscope (Carl Zeiss, Oberkochen, Germany). Photographs were taken using a digital imaging system consisting of a digital camera (Axiocam 506 clolor, Carl Zeiss, Germany) and image analysis software (Zen 2.5 blue edition, Carl Zeiss, Germany). A microscopic total damage score was determined in a blind fashion based on the presence (score = 1) or absence (score = 0) of goblet cell depletion, the presence (score = 1) or absence (score = 0) of crypt abscesses, the destruction of mucosal architecture (normal = 1, moderate = 2, extensive = 3), the extent of muscle thickening (normal = 1, moderate = 2, extensive = 3), and the presence and degree of cellular infiltration (normal = 1, moderate = 2, transmural = 3) [21 (link)].

A total macroscopic damage score was calculated for each animal as described previously [19 (link), 20 (link)]. The macroscopic scoring was based on five parameters: diarrhea (normal = 0; soft = 1; liquid = 2), presence of bleeding (no = 0; yes = 2), edema (no = 0; mild = 1; intense = 2), erythema (no = 0; mild = 1; intense = 2), and adhesions (no adhesion = 0; troublesome dissection = 1; visible adhesions = 2). Further, macroscopic tumors were counted, and the number of tumors (diameter: < 3 mm and > 3 mm) was calculated. To reduce observer bias, the whole macroscopic evaluation was designed in a blinded setup.
For microscopic evaluation, the colonic tissue was fixed in 10% formalin overnight, then routinely dehydrated and embedded in paraffin. Three 5 μm sections per colon were cut and stained with hematoxylin and eosin. Subsequently, photographs were taken using an Axio Imager A2 microscope (Carl Zeiss, Oberkochen, Germany) and a digital imaging system consisting of a digital camera (Axiocam 506 clolor, Carl Zeiss, Germany) and image analysis software (Zen 2.5 blue edition, Carl Zeiss, Germany) with 20 × magnification.

After the macroscopic damage evaluation, segments of the distal colon were stapled flat, mucosal side up, onto cardboard and fixed in 10% neutral-buffered formalin for 24 h at 4 °C. Samples were then dehydrated in sucrose, embedded in paraffin, sectioned at 5 μm and mounted onto slides. Subsequently, sections were stained with hematoxylin and eosin and examined using an Axio Imager A2 microscope (Carl Zeiss, Oberkochen, Germany). Photographs were taken using a digital imaging system consisting of a digital camera (Axiocam 506 clolor, Carl Zeiss, Germany) and image analysis software (Zen 2.5 blue edition, Carl Zeiss, Germany). A total microscopic damage score was expressed in arbitrary units (A.U) calculated based on the following parameters assessed by two researchers (AM and KP): the presence (score = 1) or absence (score = 0) of goblet cell depletion, the presence (score = 1) or absence (score = 0) of crypt abscesses, the destruction of mucosal architecture (normal = 1, moderate = 2, extensive = 3), the extent of muscle thickening (normal = 1, moderate = 2, extensive = 3), and the presence and degree of cellular infiltration (normal = 1, moderate = 2, transmural = 3) (Salaga et al. 2017 (link)).