Hematoxylin and eosin solution

Colon tissues from mice were analyzed in accordance with a previously published protocol^{59 (link)}. In short, the tissues were excised, distal colonic segments (0.5 cm) were removed and fixed with 4% neutral-buffered formalin (Hepeng Biology, Shanghai, China) for 24 h and subsequently embedded in paraffin. Cross sections of the colon (4 μm) were cut and mounted on slides. The cross sections were then stained with hematoxylin and eosin solution (Sigma-Aldrich). Tissue damage was evaluated by using a score for the severity of epithelial injury, the extent of inflammatory cell infiltration and goblet cell depletion, according to a previously described protocol^{59 (link)}. Colonic damage was assigned scores as shown in Table 1^{26 (link)}.Table 1

Colonic damage score criteria used to evaluate the severity of epithelial injury, the extent of inflammatory cell infiltration and goblet cell depletion in mouse tissue.

Score	Colonic damage	Inflammatory cell infiltration	Submucosa	Muscle/serosa
0	Normal	Normal	Normal	Normal
1	Hyperproliferation, irregular crypts, goblet cell loss	Mild	Moderate to severe	Moderate to severe
2	Mild to moderate cypt loss (10–50%)	Modest	Severe
3	Severe crypt loss (50–90%)	Severe
4	Complete crypt loss, surface epithelium intact
5	Small to medium-sized ulcers (<10 crypt widths)
6	Large ulcers (≥10 crypt widths)

Tumor fragments were placed in pathological blocks with the subsequent formalin fixation (Sigma-Aldrich, St. Louis, MO, USA), and then they were passed through alcohols to dehydrate and degrease tissues, after which the material was impregnated with paraffin. Next, sections that were 3 μm thick were made. The process of paraffin removal was carried out, sequentially passing the material through xylenes and different alcohol concentrations. After that, the slides were washed with water and immersed in hematoxylin for staining cell nuclei for 5 min (Mayer’s hematoxylin, Sigma-Aldrich, St. Louis, MO, USA). Next, the slides were placed in water to remove excess dye. After that, they were cleaned with water and stained with hematoxylin and eosin solution (Sigma-Aldrich, St. Louis, MO, USA). After carbol-xylene solution clarification, a coverslip was placed over them.
Further slides were studied with the participation of three experienced pathologists with proven competencies in the field of neuropathology who, based on pathohistological, genetic (IDH1 and IDH2 genes mutational status, 1p/19q cooperative deletion), and, if necessary, immunohistochemical parameters, made a diagnosis according to the WHO classification criteria [5 (link)].