Putt s eosin

Sections were stained with Mayer’s haematoxylin ‘Z’ stain (CellPath Ltd., Newtown, UK) for 5 min and then rinsed in tap water. Next, the sections were dipped three times in 1% acid alcohol (methylated spirit:hydrochloric acid; 100:1), rinsed in tap water, counterstained with eosin solution (1% [w/w] eosin Y:Putt’s eosin [Cellpath, Newton, UK]; 8:1) before rinsing again in tap water. The slides were dehydrated in absolute ethanol for 2 min then 1 min (twice), before being cleared with xylene (5 min) and mounted with Pertex medium (HistoLab Products Ab, Gothenburg, Sweden). Once dry, the slides were examined using an upright light microscope (BX53M; Olympus, Southend-on-Sea, UK) and images were collected with a digital camera (SC100; Media Cybernetics, Abingdon, UK) and cellSens 1.17 software (Olympus).

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.