Buffered formalin

Animals received experimental diets for a period of 28 days. Animals were observed daily and weighed weekly. Once a week, blood samples were taken from the external jugular vein of all the animals for antibody and biochemical analyses. Plasma biochemistry was performed at GenoToul-Anexplo platform (Toulouse, France) using a Pentra 400 Clinical Chemistry benchtop analyzer (Horiba, Les Ulis, France). The total concentration of the immunoglobulin subsets was measured by ELISA as previously described [46 (link)].
The ratio of the sphingoid bases sphinganine to sphingosine (Sa/So) was measured in the plasma after a regular Bligh and Dyer extraction and analysis as previously described [47 (link)].
At the end of the experiment, piglets were subjected to electrical stunning and euthanized by exsanguination. Samples of jejunum, jejunum with Peyer’s patches, colon, lung, liver, kidney, heart, spleen, and mesenteric lymph nodes were fixed in 10% buffered formalin (Sigma, Saint-Quentin Fallavier, France) for histopathological evaluation.

All transgenic mice (n = 10–12 per group) had free access to diet and water and were housed in specific pathogen-free conditions. All animal experiments were approved by the Institutional Animal Care and Use Committee at MD Anderson. Virgin female and male mice were euthanized via CO₂ at 2, 3.5, or 4 months of age. Mice's lung tissues were perfused and then fixed in 10% buffered formalin (Sigma) for ex vivo MRI and histopathological examination. Part of each fixed lung tissue specimen was embedded in paraffin, sectioned, and evaluated by a pathologist to determine the stage of the cancer. Lung tumor tissue was also collected, snap-frozen in liquid nitrogen, and stored at −80°C until further analysis.

To investigate the effects of DBT on DNCB-induced AD-like symptoms in mice, we evaluated the skin thickness. To evaluate skin thickening and mast cell infiltration, the dorsal skin samples (1 × 0.4 cm²) were obtained at the end of the experiment (on day 19). The sample was fixed in 10% buffered formalin (Sigma Aldrich, MO, USA) for at least 24 h, progressively dehydrated in solution containing an increasing percentage of ethanol (70%, 80%, 95%, and 100%, v/v), embedded in paraffin under vacuum, and sectioned at 4 μm thickness. Deparaffinized skin sections were stained with hematoxylin and eosin (H&E) for skin thickening and toluidine blue for mast cell infiltration. Histopathological changes were examined using the Leica Application Suite (LAS; Leica Microsystems, Buffalo Grove, IL). The magnification was ×100. The epidermal thickness was measured from the top layer (stratum corneum) to the bottom layer (stratum basale). The dermis thickness was measured in vertical distance between stratum basale layer and the subcutaneous tissues. Thickness was measured 3 times at regular intervals in one slide and obtained 15 results per group [21 (link)]. The number of mast cells was measured in the entire area of slides for each sample (n = 5).

Tumor-bearing mice were sacrificed after PET imaging and tumor were excised, fixed in 10% buffered formalin (Sigma, Saint Louis, MO) and embedded in paraffin. For histopathological analysis, tissues were serially sectioned (3 μm) and stained with hematoxylin and eosin (H&E). For analysis of MT1-MMP expression, immunohistochemical staining was performed using anti-MT1-MMP LEM 2/15 antibody at 1:400 dilution after antigen retrieval with low pH buffer in Autostainer platform (Dako, Santa Barbara, CA) and counterstained with hematoxylin. Whole slides were acquired with a slide scanner (AxioScan Z1, Zeiss, Jena). Regarding analysis, an appropriate script was created using Zen Blue Software (V 3.1, additional module for analysis, Zeiss, Jena).