I am preparing to do cell senescence staining recently. The fixative in the kit is almost used up, and the lab just happens to have PFA and formalin. The kit instructions say that the fixative is paraformaldehyde, but the concentration is not clearly marked (it seems that the formulas of different brands are different?).
I am a little worried that direct replacement will affect the staining effect-for example, does the fixation time need to be adjusted? Will it cause β-galactosidase inactivation, or will changes in cell morphology affect the staining results? Has anyone tried to use PFA/formalin to replace the fixative in the kit?
If it is feasible, what concentration and processing time are recommended? What details need to be paid attention to to ensure the success rate of staining? Welcome to share your experience or avoidance guide, thank you!
[Troubleshooting Questions are selected from MCE customer consultation emails.]
Dear Dr. Lucian Miles,
No, you should not use PFA/formalin as a fixative. Though PFA is a common fixative, it can react with and inactivate beta-galactosidase, making it unsuitable for staining where enzyme activity needs to be preserved.
Instead, use Glutaraldehyde, especially at lower concentration (e.g., 0.2% in PBS) for 30 minutes. It is a better choice for preserving enzyme activity during fixation. It will allow for the localization of beta-galactosidase by maintaining its enzymatic activity, unlike many other aldehydes.
As far as I can recollect one of the senescence beta- galactosidase staining kit uses 2% formaldehyde, 0.2% glutaraldehyde, and 1X PBS when diluted to a 1X final solution as the fixative. You may try this as well.
I have provided you with the concentration and incubation time, but you may have to optimize these parameters for better results.
Regards,
Malcolm Nobre
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