Tissue tek optimum cutting temperature compound

Samples of mid-ileal tissue were collected at necropsy and rinsed with Dulbecco's phosphate buffered saline (D-PBS) at pH 7.4 (Sigma-Aldrich, St. Louis, MO). The luminal side was placed on top of a liver section with a thin layer of Tissue-Tek optimum cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA) interface to protect microvilli structure from mechanical and environmental disruption. This also aided in determining tissue orientation upon cryosectioning. Tissue samples were then wrapped in aluminum foil and snap-frozen by placement in a tin cup of cold isopentane (Sigma-Aldrich) housed within a small cooler of dry ice and 95% ethanol slurry for a minimum of 5 min, then wrapped again in aluminum foil and transferred on dry ice to be stored at −80°C.
When cryosectioning was ready to be performed, tissue sections were removed from storage at −80°C and placed in a cryostat to acclimate to −20°C for at least 30 min. The entire tissue sample was embedded within Tissue-Tek optimal cutting temperature (O.C.T.; Sakura Finetek, Torrance, CA) and cut in 6 μm sections, then adhered to MAS slides (Matsunami Glass, Bellingham, WA). The tissue sections were dried overnight at room temperature and fixed in a 50% acetone/50% methanol solution for 5 min. Slides were stored at −80°C until ready for further processing.

After collection, the maxillae and colons were fixed in 4% PFA. The colons were then paraffin-embedded and sectioned, followed by H&E staining. At the same time, the maxillae were decalcified in 0.5 mol·L^–1 EDTA for 3 weeks, dehydrated in a 30% sucrose solution and embedded in Tissue-Tek optimum cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA). They were cut with a freezing microtome (Leica CM1900, Germany) and stained with H&E (Servicebio, Wuhan, China) as well as tartrate-resistant acid phosphatase (TRAP, 387A-1KT, Sigma-Aldrich). The distance between the cementoenamel junction and alveolar bone crest (CEJ-ABC distance) of the sections stained by H&E was measured to evaluate bone loss. TRAP-positive multinucleate cells were considered osteoclasts and a sign of bone resorption.
For assessment of colon inflammation, histological scores were determined by the following criteria: the severity of epithelial/crypt loss (score, 0-4) and the extent of inflammatory cell infiltration in the lamina propria (score, 0-4). Each score was multiplied by a factor representing the percentage of the colon involved (1, 0%–25%; 2, 26%–50%; 3, 51%–75%; 4, 76%–100%), and the scores on the individual measures were then added to calculate the overall histological score for each sample, as previously described.^{41 (link)}