Surgipath 10 neutral buffered formalin

Harvested colonic tissue was fixed in Surgipath 10% neutral buffered formalin (Leica Biosystems, Concord, Ontario, Canada) for 24 h. Formalin-fixed tissues were dehydrated in ethanol and placed in Histo-Clear (Diamed Lab Supplies, Mississauga, Ontario, Canada) prior to embedding in paraffin at 60°C. Sections (5 µm) were deparaffinized with xylene and stained with hematoxylin and eosin (H&E). Tissues were scored for mucosal damage by an experienced veterinary pathologist blind to the treatments using an established scoring guide (34 (link)) that ranked common characteristics of mucosal damage at scores from 0 to 4, with 4 representing pronounced damage and 0 representing minimal to no damage (see Table S1 in the supplemental material). Sections were also scored (0 to 3 or 4) for epithelial cell wall hyperplasia based on a mild to severe increase in the number of cells found within crypt columns; for crypt height based on minimal to maximal increases in height; for epithelium cell injury (noting the degree of focal erosions and cell shedding); for the degree of inflammation based on the number of neutrophils and mononuclear cells present in the lamina propria; for goblet cell depletion based on the number of goblet cells and mucin droplet size; and for the degree of mitotic activity based on how much of the epithelial cell displayed increased activity (Table S1).

On days 14, 21 and 28 p.i., arbitrarily selected mice from each treatment were anesthetised with isoflurane and euthanized by cervical dislocation under anesthesia. To visualize the intestine and collect samples, a mid-line laparotomy was used to exteriorize the intestine [65 ]. A gross pathological assessment of the large intestine with photo-documentation was completed, and the length and width of the cecum and colon were measured. The cecum and colon were longitudinally incised, and luminal contents were collected and stored at −20 °C for DNA extraction and characterization of the microbiota. Sections of the distal colon were collected and immediately placed in RNAlater™ (Qiagen Inc., Toronto, ON) and subsequently stored at −20 °C for mRNA extraction. Sections from the proximal and distal colon were collected and frozen for subsequent for DNA analysis. Samples of histological examination were placed in Surgipath^® 10% neutral buffered formalin (Leica Biosystems, Concord, ON) for histopathologic scoring, and in Methacarn (60% methanol, 30% chloroform, 10% glacial acetic acid) for mucus analysis [90 (link)].

Formalin-fixed paraffin-embedded (FFPE) human breast cancer tissues were obtained from the Department of Anatomical Pathology, Division of Pathology, Singapore General Hospital (SGH). The present study utilized archival specimens from a cohort of 388 TNBC breast cancer patients diagnosed at the Division of Pathology of SGH from 2003 to 2015. The Centralized Institutional Review Board (IRB) of SingHealth provided ethical approval for the use of patient material in this study (CIRB ref: 2018/2910-2013/664/F). Clinicopathological parameters including age, ethnicity, tumor size, histologic grade, subtype, associated ductal carcinoma in situ, lymphovascular invasion, axillary lymph node status, tumor borders, and growth patterns were reviewed.
The tissues were fixed and processed in Surgipath 10% neutral buffered formalin (Leica Biosystems, Wetzlar, Germany) for 6–48 h in accordance with the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) Clinical Practice Guidelines [41 , 42 ].