GFP bleaching

[chenmak]

I have cells transfected with Luciferase-GFP, which are part of my tissue I am staining (brain tumor tissue).
Does anyone have an experience if it bleached and what is the best bleaching solution to use?

Thank you

Chen

[radtkea]

Hi Chen,

Thank you for your question! Sorry to answer you so late in the forum after answering your private email.

In our original PNAS 2020 paper, we reported that CFP, GFP, YFP, and RFP did not bleach after 10 rounds of LiBH4 treatment.

These details are also found in the Fluorescent Probes Tested link on the IBEX Imaging Community site.

However, the MacDonald and Chu labs report elimination of GFP after treatment with antigen retrieval by boiling slides in sodium citrate solution (pH 6) for 20 min. This treatment inactivated the fluorescent protein. They used an anti-GFP primary and Alexa Fluor secondary antibodies to detect the GFP protein after antigen retrieval.

@colinchu72 Do you have a detailed protocol that you can kindly share with us?

We did explore the use of UNI-TRIEVE, a more gentle antigen retrieval method for fixed frozen tissues. The idea was that this gentler protocol would be more compatible with fixed frozen tissues and wouldn't destroy protein epitopes. Unfortunately, the results were inconclusive with RFP, and the AR buffer prevented previously validated antibodies from labeling the treated tissues.

Please keep us posted!
Andrea