4.3. Clinical Observations and Histopathological Analysis

We observed the mice on a daily basis for clinical signs and sacrificed them after 28 days. We measured body weight every three days. We collected blood to evaluate hematological parameters under anesthesia at the end of the treatment period. The tumor specimens were divided into two groups. The first group was fixed with 10% formalin and embedded in paraffin. These specimens were then assessed using hematoxylin and eosin staining; the terminal deoxyribonucleotidyl transferse (TdT)-mediated biotin-16-dUTP nick-end labeling (TUNEL assay; APO-BrdU™TUNEL Assay Kit, BDPharmingen, San Diego, CA, USA); and immunohistochemistry (IHC) including IL-1β, TNF-α, CD44, EGFR, and TGF-β. We used IHC staining to assess for IL-1β, TNF-α, CD44, EGFR, and TGF-β in the tumor using primary antibodies against IL-1β, TNF-α, CD44, EGFR, and TGF-β (Merck, Billerica, MA, USA). We measured protein expression through IHC using the TAlink mouse/rabbit polymer detection system from BioTnA (Kaohsiung, Taiwan). We employed Moticam 2300 (Motic Instruments, Richmond, BC, Canada), a high-resolution digital microscope equipped with Motic Images Plus (version 2.0) to capture images and analyze adipocyte size distributions between the control and treated obese mice. The other group was preserved in a freezer at −80 °C and the levels of caspase 3, ERK, Bcl-2, COX-2, and VEGF were examined using Western blotting.