Zen 2.5 blue edition

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)).

Five days old T3 double transgenic seedlings were further analyzed by confocal microscope LSM700 (Carl Zeiss, Germany) with Argon laser source fitted with Plan-Apochromat 20×/0.8M27 objective lenses. GFP and FM4-64 and mCherry were excited at 488 and 555 nm, respectively. Colocalization analysis was done using Zen 2.3 SP1 FP1 (black) software (Carl Zeiss, Germany) under the default setting. Image analysis was performed using Zen 2.5 (Blue edition) (Carl Zeiss, Germany) and ImageJ⁵.

Microscopic analyses were performed with the Zeiss Cell Observer SD, a confocal spinning disc microscope, and a 63x/1.4 oil objective lens (Carl Zeiss Microscopy, Jena, Germany) using 488 nm-, 506 nm-, 587 nm- and 592 nm laser lines. For mitochondrial membrane potential analysis, TMRM staining solution (1 µM tetramethylrhodamine methyl ester perchlorate; Sigma-Aldrich, St. Louis, MO, USA; T5428; dissolved in H₂O) was incubated for 30 min. For the staining of lipid droplets, freshly grown mycelium was incubated with LipidSpot™ 488 (Biotium, Fremont, CA; 70065) for 15 min. For the analysis of vacuoles, freshly grown mycelium was treated for 5 h with 2 μg/mL FM™ 4-64 Dye (Invitrogen, Waltham, MA, USA; T13320) before microscopic analysis. For image processing, the Zeiss microscopy software ZEN 2.5 (blue edition) was used. The size and distribution of vacuoles, as well as the intensity of TMRM, were measured manually using ZEN software.