2.4.3.2 Procedure

2.4.3.2 Procedure

The mid-section of the right femur of the extracted mouse is immersed in 4% neutral formaldehyde for 10 hours, the bone tissue is cut to a thickness of no more than 0.5 cm, and placed in the decalcification solution for 15 hours, changing the solution every 5 hours. Once the bone can be pierced with a needle, it is removed, neutralized with 5% sodium sulfate solution for 2 hours, and rinsed with running water for at least 24 hours. The bone tissue blocks are then sequentially placed in 80% alcohol for 15 minutes, 90% alcohol for 15 minutes, and twice in 100% alcohol for 30 minutes, followed by immersion in an 80% alcohol–xylene mixture for 10 minutes, xylene for 10 minutes for dehydration, and waxing for 1–2 minutes. A hard wax block is prepared, a small portion of wax is melted in the middle of the block with hot tweezers, and then the already paraffin-saturated tissue block is taken out of the wax cup with hot tweezers and dropped into the molten wax. After cooling and solidifying, paraffin sections are immediately made. Each specimen is made into a 4 μm thick tissue section, with 3 consecutive sections made for each specimen. After the paraffin sections are affixed, they are briefly baked over a flame to melt all the wax on the tissue sections and dry any moisture, then dipped in xylene, 100% alcohol, and 95% alcohol for 30 seconds each to remove the wax, rinsed with water, and stained with HE-Giemsa for future use.

2.5 Testing Methods and Indicators