Safranin o staining kit

At the termination of the vivo experiments, rats were euthanized with 1.5% pentobarbital sodium (30 mg/kg rat body weight). The target discs of the caudal vertebrae were harvested for further analysis. Then the samples were fixed in 4% paraformaldehyde for 1 day and decalcified in 14% Ethylene Diamine Tetraacetic Acid (EDTA) for 30 d. Subsequently, discs were embedded in paraffin (Leica, Richmond, VA, USA), and cut into 5-μm sections in the coronal plane using a microtome (Leica, Heidelberg, Germany). Hematoxylin & Eosin (H&E) staining was performed to examine tissue histology. Briefly, the sections were stained with hematoxylin solution for 5 min and incubated in 1% acid ethanol (1% HCl in 70% ethanol). The sections were washed with pure water. Afterward, the sections were stained with an eosin solution for 3 min, dehydrated with graded alcohol and cleared in xylene. Safranin O-Fast green (S.O.) staining was performed to determine changes in proteoglycans. The sections were stained with a safranin O staining kit (Solarbio, Beijing, China) according to the recommended procedure from the manufacture.

Samples originating from ex vivo-cultured murine IVD tissues and human samples were dissected and fixed in 4% paraformaldehyde for 3 days. After decalcification in 10% EDTA (pH 7.2-7.4), the samples were processed, embedded in paraffin and cut into 5-μm sections using a microtome. Standard H&E staining was used to examine tissue histology. Briefly, the sections were stained with a haematoxylin solution for 5 min and incubated in 1% acid ethanol (1% HCl in 70% ethanol). The sections were washed with pure water. Then, the sections were stained with an eosin solution for 3 min, dehydrated with graded alcohol and cleared in xylene. Safranin O and fast green staining was performed to determine changes in proteoglycans. The sections were stained with a Safranin O staining kit (G1371, Solarbio) according to the manufacturer's recommended procedure.