P53 clone do 7

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)].

Routinely-processed, formalin-fixed and paraffin-embedded tissue blocks were selected and 5-μm serial sections were prepared from the cut surface of the blocks. The antibodies, MUC1 (clone Ma695), MUC2 (clone Ccp58), MUC5AC (clone CLH2), MUC6 (clone CLH5; 1:200 dilution; Novocastra, Milton Keynes, UK), claudin-3 (polyclonal), claudin-4 (polyclonal), claudin-18 (polyclonal; 1:50 dilution; Zymed Laboratories, South San Franscisco, CA, USA), Cdx2 (clone CDX2–88; 1:500 dilution; BioGenex Laboratories, Freemont, CA, USA), p53 (clone DO-7; 1:100 dilution; Dako, Carpinteria, CA, USA) and Ki-67 (clone MIB-1; 1:100 dilution; Immunotech, Marseille, France) were used. Immunoperoxidase reactions were performed using the Ventana BenchMark^® LM automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s instructions. All cases were reviewed by two investigators, who arrived at a consensus on the pathological diagnoses and the assessment of immunoreactivity.

IHC was performed using the Bond Polymer Refine Detection kit (Leica Biosystems, Newcastle upon Tyne, UK) in the BOND-MAX system (Leica Biosystems). Four-micrometer-thick FFPE sections were incubated with the following primary antibodies: PMS2 (clone EP51; dilution: 1:50 Dako), MSH6 (clone EP49; dilution: 1:50; Dako), E-cadherin (clone NCH-38; dilution: 1:50; Dako), and p53 (clone DO-7; dilution: 1:150; Dako). IHC slides were jointly evaluated by three pathologists (VA, GP, and MF).
Mismatch Repair defective status was assessed by testing PMS2 and MSH6, and samples were defined as dMMR when one or both proteins resulted negative [15 (link)]. In case of protein loss, the dominant component of the heterodimer (i.e., MLH1 for PMS2 and MSH2 for MSH6; Dako) was tested.
p53 was considered as aberrant in the presence of complete loss or diffuse and strong nuclear immunostaining in dysplastic cells [9 (link), 16 (link)].
E-cadherin expression was considered altered in the presence of complete loss or markedly reduced membranous staining (> 30%), regardless of nuclear/cytoplasmic staining [9 (link)].

ER, PR and p53 protein expression was assessed by immunohistochemistry. Selected FFPE blocks were sectioned at 3 μm thickness. Sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was achieved by incubating the sections in preheated EDTA buffer (pH 8) for ER-alpha and in citrate buffer (pH6) for PRA and p53 in a water bath (98°C) for 20 minutes. Endogenous peroxidase was blocked and the slides were incubated for 1h using the following antibodies: ER alpha (clone 6F11, mouse monoclonal antibody, 1:100, Novocastra Leica Biosystems), PRA (clone 16, mouse monoclonal antibody, 1:150, Novocastra Leica Biosystems) and p53 (clone DO7, mouse monoclonal antibody, 1:200, Dako). Finally, sections were incubated with Poly-HRP-GAM/R/R IgG (ImmunoLogic) and counterstained with hematoxylin. Appropriate positive and negative controls were also included in each assay.
ER-alpha, PRA and p53 expression was evaluated by optical microscopy using an image analysis program (GenASIs Go-Path). Fifteen random fields of the tumor in each slide (corresponding to a mean of 7138 tumors cells per case) were photographed. In each photo tumor cells were selected and evaluated using H-score. H-score was defined by the intensity grade of staining (0–3) multiplied by the percentage of positive cells, resulting in a range of possible scores of 0–300.