Why is PBS added in the final step of the Alizarin Red staining protocol, and should it be aspirated before analysis?

When assessing osteogenic differentiation of MSCs by Alizarin Red staining, the addition of PBS in the final step serves an important purpose.

Reason
PBS provides a buffered environment that enhances the precipitation of Alizarin Red, resulting in a more intense and stable staining signal.

Handling recommendation

• Do not aspirate the PBS before analysis.
• Leave the PBS on the cells and analyze the sample immediately after staining and washing.

Prolonged storage without embedding may cause the Alizarin Red dye to bleed, which can negatively affect staining quality and data interpretation.