Hello, I am trying to fix Pseudomonas aeruginosa PAO1 cells with 4% formaldehyde for 20-30 minutes (on ice and in the dark) for fluorescent microscopy. I am attempting to stain the DNA and outer membrane, but have not been successful with this fixation method. The dyes give weak signals and appear to be exported out, suggesting the bacteria are not completely fixed with formaldehyde. Does anyone have a reliable protocol for PAO1 fixation, including concentration, time, quenching, etc.? Any help would be greatly appreciated.
Not a total answer to your question, but 4% formaldehyde is known to sometimes cause loss in fluorescence signal
https://www.bio-techne.com/applications/flow-cytometry/flow-cytometry-troubleshooting-guide#:~:text=Certain%20proteins%20can%20be%20more,formaldehyde%20may%20be%20more%20appropriate.
This page may help to some degree.
I usually fix in a solution of 0.8% PFA and 0.2% Glutaraldehyde for around 15 minutes at RT for immunofluorescence and seem to get plenty of signal on T cells, not sure if this will be applicable for you.
Thank you so much Edward Sanders for your valuable suggestions to my question.
I will definitely try to fix my PAO1 cells with 0.8% PFA and 0.2% Glutaraldehyde solution for 15 minutes at RT for my next trial. I hope it provides the desired signal in my experiments as well.
Additionally, have you ever used any bacterial strains for your immunofluorescence experiments? I would be interested to know if you have any experience or suggestions regarding that.
Unfortunately all of my work has been on mammalian lines.
Good luck with the fixation
- Badges
- Science topic
- Similar topics
- Comment