Haematoxylin and eosin h e staining solution

Cell culture studies were performed using the A549 (human lung epithelial carcinoma) cell line (ATCC, USA) which was provided as a kind gift by Prof. Alaina Ammit, at the Woolcock Institute of Medical Research, Sydney, Australia. A humified 37 °C incubator was used to grow the cells. The cells were cultured in 5% CO₂ incubator using Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin, and streptomycin. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), crystal violet, dimethyl sulphoxide (DMSO), haematoxylin and eosin (H&E) staining solution were procured from Sigma-Aldrich, St. Louis, MO, USA. The human XL oncology array kits (R&D systems, USA) were purchased from in vitro technologies pvt ltd, Australia. Additional chemical reagents and consumables were obtained from Sigma-Aldrich unless specified.

We fixed the right femurs in 4% buffered formalin for 24 hours and placed in 9% formic acid for decalcification for 21 days. The sample was cut in the middle at a mid‐sagittal plane and embedded in paraffin. Samples were cut at 7 μm thickness. Slides were placed in Xylene to eliminate paraffin at room temperature, following with the dispose with graded ethanol and distilled water. Haematoxylin and Eosin (H&E) staining solution (Sigma) was used for the staining.