4.9. Bone TRAP Staining for Osteoclasts Observation

Rat femoral tissue sections were put in xylene solution for 8 min, and then put it in 100% ethanol, 95% ethanol, 80% ethanol (5 min each), the they were washed in deionized water for 3 min, stained with TRAP staining solution (pH 5.0–5.2) at 37 °C in the dark for 3 h, then the slides were washed thoroughly with distilled water and stained with hematoxylin for 1 min, after then, washed the slides with distilled water, then the slices are gradually dehydrated by gradient ethanol (80%, 95% I, 95% II, 100% I, 100% II). Then observe the morphology of femoral osteoclasts under an optical microscope. Randomly select five non-overlapping fields of view at 400× in each slide, the number of osteoclasts in each field of vision was accurately counted.