Greetings,
I have fixed MC3T3 cultures and incubated with 40mM alizarin red (pH 4.2) for 12 minutes. When rinsed with water, three times for 5 minutes each, the final wash still remains darkly coloured, indicating that there is more background alizarin to be removed. Has anyone encountered this, and do you continue washing or simply assume the background will be equivalent with equivalent washing times for all wells?
Thanks
Hi Michael, maybe you can decrease the concentration of the dye. I used 4 mM alizarin red for 5 min at RT. And then wash as many times as you think the background color is acceptable. I usually did three times. It worked for many cell lines and if the cells mineralized, the red color will not be washed away. Good luck!
Hi Michael, try also to wash 3 times with distilled water several times (at least 3 times) after fixation, before adding alizarin red. With Von Kossa staining we are used to do in this way to avoid background
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