Hi friendly people!

  I've been doing live imaging of MDCK epithelial migration and I've noticed extreme variability in Hoechst staining. I use the NucBlue formulation where you just add 4 'drops' of staining solution to media and incubate the cells for 30 minutes at 37 degC before changing media and imaging. For several weeks, this has worked remarkably well and allowed live nuclear imaging during overnight experiments with low exposures/intensity and no apparent bleaching. However, I recently thawed a new batch of cells that look great in every way except that the Hoechst stain vanished over the course of an overnight run. As far as I know, I haven't changed my protocol, so I'm wondering if there's something important about Hoechst staining that I'm not aware of. 

Anyone have a similar experience?

Thanks!

Daniel

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