Hello,

I am conducting an experiment to induce osteoblast differentiation using MC3T3-E1 cells. Cells are treated with differentiation reagents for 7 days. After treating the cells with the differentiation reagent, I confirmed differentiation through qPCR analysis of differentiation markers. However, I encountered an issue with Alizarin Red staining.

After washing the cells with PBS, I fixed them with methanol, paraformaldehyde, or neutral-buffered formalin. However, I couldn't observe any difference between the control and treated groups.

I've attached a picture for your reference. Could you please provide any suggestions for improving this experiment?

Alizarin Red solution is prepared at 2% in distilled water and adjusted to a pH of 4.18.

Thank you