Is there anything special to keep in mind while fixing Arabidopsis roots expressing fluorescently labelled proteins (e.g.-Tub-RFP, VHaA1-GFP) to ensure their structure, localization and fluorescence are not hampered? I usually use the widely used protocols utilizing Formaldehyde and Glutareldehyde followed by methanol and NaBH4 treatments.
Thanks
Check the protocols for tissue fixation using methacrylate to see your reporter proteins such as GFP or another GFP-derived reporter protein.
Regards
Hi Rahul,
It seems you look for alternatives to formaldehyde based protocols but I have recently PFA- fixed those same markers and they look pretty bright.
For fixation we use Paraformaldehyde 4% in 50mM PIPES, 5mM EGTA, 5mM MgSO4, pH 7). We mount the fixed roots on poli-Lysine coated slides. Once mounted we add a drop of a Glycerol-based fluorescence antifade reagent named Citifluor AF1. We keep the slides at 4C in the dark and fluorecence last for months.
Hope it helps.
Abel
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