Tissue tek medium

After decapitation of anesthetized mice, the liver was removed and fixed using a buffered 10% formalin solution (pH 7.4) for 24 hours, and then the liver tissue was washed with 0.1 M Na⁺-phosphate buffer, pH 7.4. To prepare the liver sections the cryoprotection procedure was used. The liver tissue was immersed in a buffered solution containing 30% sucrose (+4°C, 48 h), and then frozen using dry ice and Tissue-Tek medium (“Sakura Finetek Europe”, Netherlands). Freezing liver was cut on a cryostat (“Leika Microsystems”, Germany), and the sections (7 μm thickness) were mounted on the Super Frost/Plus glasses (“Menzel”, Germany). The liver sections from animals of each of the studied groups were mounted on the same glass. According to standard histological procedures, the sections were stained with hematoxylin and eosin (“Labiko”, Russia), and after treatment with alcohol and xylene were placed into the BioMount medium (“Bio Optica”, Italy). In the case of the Sudan staining, the liver sections were washed sequentially with distilled water and 50% ethanol and then immersed in a solution of Sudan III (“Labiko”, Russia) for 15 min. After washing with distilled water and 50% ethanol, the Sudan-stained liver sections were stained with hematoxylin, washed with distilled water, placed under a cover glass using glycerol and used for histochemical analysis [53 ].

Biopsies were obtained from Mexican women seen at Hospital IMSS “La Raza” (Mexico City) and “Hospital Juárez de México” (Mexico City) and who were pathologically diagnosed with Atypical Squamous Cells of Undetermined Significance (ASCUS), Low-grade Squamous Intraepithelial Lesion (LSIL), High-grade Squamous Intraepithelial Lesion (HSIL), or Cervical Cancer (CC). The present study conducted according to the Declaration of Helsinki for Medical Protocol and Ethics, and the Institutional Committee of Research and Ethics approved the study (registration no. HMJ 2231/13-B). Expert colposcopists performed all examinations, and samples were classified according to the International Federation of Gynecology and Obstetrics (FIGO) [11 (link)]. Fifty-five biopsies of the cervical lesions from patients with an age range between 20 and 65 years (8 ASCUS, 25 LSIL, 11 HSIL, and 11 CC) plus 25 biopsies of adjacent regions without morphological changes, for a total of eighty samples, were analyzed.
Biopsies were placed in Tissue-Tek medium (Sakura Finetek, USA) for their preservation and stored at −80°C prior to DNA extraction.

A total of eight animals (four per group) were used for the immunohistochemistry analyses. The small intestine segments were transferred to Tissue‐Tek® medium (Sakura Finetek), frozen in liquid nitrogen, and sliced into 6‐μm‐thick sections. We performed the immunohistochemistry technique using the Mouse and Rabbit Specific HRP/DAB IHC Micro‐polymer detection kit (ab236466, Abcam), following the manufacturer's instructions. The slides were blocked with 3% BSA and incubated overnight with rabbit anti‐IL‐1β antibody (ab9722, Abcam) diluted 1:250. The antigen–antibody complex was detected using chromogen 3,3′‐diaminobenzidine (DAB). Sections without the primary antibody (IL‐1β) were used as a negative control for the immunolabeling process. This qualitative evaluation was employed to identify the enteric immunoreactive cells. Photomicrographs were captured with an Eclipse® microscope (Nikon) equipped with a 40× objective.