Immunohistochemical Evaluation of Tongue Tumors

The volume of the animal tongues tumor was recorded after the experimental period and were measured based on the formula (length × width × height × π/6) as mentioned in a previous study [23 (link)]. The tongue sections were analysed histologically and classified as squamous cell carcinoma, dysplasia, hyperplasia, or normal, per animal [5 (link)]. Immunohistochemical evaluation was conducted in the present study to determine the effect of the FD extract, the expression of tumor markers, cyclin D1, bcl2, p53, β-catenin, and e-cadherin in the tongue tissue sections of the Sprague-Dawley rats. Formalin-fixed, paraffin-embedded tissue sections (4 μm) were cut from the paraffin blocks and deparaffinized and rehydrated. The antibodies were then applied according to the manufacturer's protocol at the pathology laboratory, Pathology Department, University of Malaya. Immunohistochemistry was conducted using the Ventana Benchmark XT auto stainer with the following antibodies: p53 (clone DO-7, Dako Japan), e-cadherin (clone NCH-38, Dako Japan), β-catenin (clone b-Catenin-1, Dako Japan), bcl2 (clone 124, Dako Japan), and cyclin D1 (clone SP4, Thermo Fisher Scientific). The IHC staining was carried out based on the automated process for routine staining at the pathology laboratory. The IHC computed analysis using Image J software was as explained in our previous work [24 (link)].