Haematoxylin

Paraffin-embedded tumor samples were sectioned (4 μm) and mounted on microscopic slides. Heat-induced epitope retrieval was performed in 10 mmol/L citric acid buffer at pH 7.2 preheated in a microwave and the sections were heated at 98 °C for 20 min. Sections were incubated with the primary antibody Ki67 (DAKO M7240, clone MIB-1, 1:250) at 4 °C overnight, rinsed with TBST, and incubated for 1 h at room temperature with biotin-conjugated secondary antibody, followed by 30 min incubation with ABC complex (VECTASTAIN^® ABC Kit, Vector Laboratories, Burlingame, CA, USA). Visualization was achieved using DAB chromogen (DAKO K3468) as the substrate, and slides were counterstained with haematoxylin (CellPath Ltd., Newtown, UK) and mounted with Entellan^® new (Merck Millipore, Burlington, MA, USA, 107961). Images were obtained using a Nikon TE2000 inverted microscope (Nikon Instruments Inc., Melville, NY, USA), and the percentages of Ki-67 positive cells were calculated, counting positive and negative cells using the point tool in the ImageJ freeware.

The chemicals used for this study included, methanol, glacial acetic acid, ammonia, ascorbic acid and chloroform all purchased from HmbG Chemical, Malaysia. Others includes, n-hexane and ferric chloride hexahydrate (FeCl₃.6H₂O) purchased from Bendosen, Malaysia. 2-diphenyl-1-picrylhydrazyl (DPPH) was procured from Sigma, Switzerland while sodium acetate, xanthine, xanthine oxidase and allopurinol were purchased from Sigma-Aldrich, Germany. Additionally, sodium dihydogen phosphate (NaH₂PO₄), disodium hydrogen phosphate (Na₂HPO₄), carrageenan, Tween 20, aspirin, sodium chloride, Dragendorff's and Mayer's reagent were purchased from R&M chemical, Malaysia. Haematoxylin, putts eosin and DPX were purchased from Cellpath, UK, hydrochloric acid (HCl), formaldehyde, xyleneand ethanol were purchased from Systerm, UN. Finally, 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) (Acros, US), ketamine (Ketaset®), xylazine 100 mg/ml (Rompun®), ferrous sulphate heptahydrate (FeSO4.7H₂O) (Fisher chemical, US) and paraffin wax (LEICA, Germany) were purchased from the respective sources.