I use 2% PFA (in PBS) for 30 min at room temperature with my mCherry expressing cells without any problems. I could also see mCherry fluorescence even after one week without any problems. Good luck

Hi Noelle,

I have been examining the expression of an mCherry construct recently in neurons. 4% PFA fixation (in PBS, pH = 7.4) for 10 minutes at room temperature should be adequate for visualising the fluorescence. Just keep the sample in the dark as much as possible during fixation and subsequent handling.

Best,

James

hi

Thank you very much for yours answers, i will test them on friday on my cells.

have a nice day

Noelle

There are many available aldehydes they work across a range of treatment time. Try Glutaraldehyde !!

for more information refer following papers:

• Rolls GO, Farmer NJ, Hall JB. Artefacts in histological and cytological preparations. In Woods A and Ellis R eds. Laboratory histopathology. New York: Churchill Livingstone, 1994;5.3-1 - 5.3-29.
• Eltoum I, Fredenburgh J, Myers RB, Grizzle WE. Introduction to the theory and practice of fixation of tissues. J Histotechnol 2001;24;173 -190.
• Eltoum I, Fredenburgh J, Grizzle WE. . Advanced concepts in fixation: 1. Effects of fixation on immunohistochemistry, reversibility of fixation and recovery of proteins, nucleic acids, and other molecules from fixed and processed tissues. 2. Developmental methods of fixation. J Histotechnol2001;24;201-210.
• Hope that helps. Thank you!!

Hello everyBody,

thank you for your answer, i try PFA and FA1% final and it work very well!!!.

I keeped my cells in dark and i made the cytometry FACS the same day to avoid decrease or extinction of fluorescence.

Thank you very much for your help!

Have a nice day!!:)