To begin, prepare wax blocks from euthanized mice injected with Xiaoyao pills. Cut the wax blocks into six micrometer thick continuous sections, and bake the slices at 70 to 72 degrees Celsius for 30 to 60 minutes. Transfer the slices to the de-waxing solution for eight minutes, then dehydrate twice in 75%anhydrous ethanol for five minutes each before rinsing in PBS for five minutes.
Now perform EDTA high pressure repair for 15 minutes. After rinsing with PBS, dropwise add 3%hydrogen peroxide to the tissue and incubate for 10 minutes. Wash the tissue three times with PBS for five minutes each.
Using an immunohistochemistry pen, draw a circle around the tissue. After washing the tissue with PBS, stain overnight in primary antibody solution. The following day, rinse the tissue three times for five minutes each in PBS.
Dropwise add the secondary antibody and incubate for 60 minutes. After rinsing with PBS, incubate the tissue in DAB ready-to-use solution for three to five minutes. Now stain the tissue in hematoxylin for three minutes.
Again, rinse the tissue with PBS before placing it in differentiation solution for 10 seconds. After PBS rinse, add the return solution for 10 seconds. Finally, perform 75%anhydrous ethanol treatment for five minutes and seal the slide with neutral resin.
Hematoxylin-eosin staining showed tightly-arranged trabeculae in sham mice, while OVX mice displayed typical osteoporotic changes with sparse trabeculae and enlarged marrow cavities. Micro CT comparison showed incomplete trabecular meshwork and thinning of the bone cortex in OVX mice compared to sham mice. After drug intervention, hematoxylin-eosin staining showed restored trabeculae structure and reduced marrow cavity compared to the OVX group.
Imaging indices showed fewer fractured trabeculae and restored reticulation in the high-dose Xiaoyao pills group compared to the OVX group. Immunohistochemistry results showed increased alkaline phosphatase and collagen type one and decreased interleukin 17, ACT1, and interleukin 6 in the drug group.
