Alizarin Red Staining of Meniscus and Osteoblasts

For Alizarin Red staining of clinical meniscus tissue sections, the clinical meniscal tissues were routinely dehydrated and embedded in paraffin (clinical meniscus samples were not decalcified). Tissue Section (4 μm) were then prepared according to standard procedures, and staining was performed after deparaffinization. The sections were immersed in Alizarin Red S staining solution (Solarbio, G3280) for 5 min, thoroughly washed with distilled water, and counterstained with hematoxylin to stain the nuclei. After routine dehydration and clearing, neutral resin was used to seal the sections. The stained sections were then observed and photographed under a microscope.
For Alizarin Red staining of calcified nodules after osteoblast induction, the Alizarin Red S Staining Kit for Osteogenesis (Beyotime, C0148S) was used. After the completion of primary meniscal cell differentiation, the cells were fixed for 20 min and washed three times with PBS. Subsequently, Alizarin Red S staining solution was added to evenly cover the cells, and the cells were stained at room temperature for 30 min. After thorough washing with distilled water, the stained cells were observed and photographed under a microscope and camera. For Alizarin Red quantification, 10% cetylpyridinium chloride solution was added to each well, and the samples were shaken slowly on a shaker for 30 min. After dissolving Alizarin Red, the absorbance was measured at 570 nm to quantify calcification.