I’ve been working on imaging cholesterol localization in human endothelial cells using Filipin III. Because the excitation for Filipin is around 350, we have to use Two-photon excitation because no one on campus has a confocal capable of such short wavelengths. It has been going well, except lately I’ve been having odd, inconsistent imaging over time.
Despite all samples being prepped at the same time with the same reagents, there is significant variation that I do not believe is real. Additionally, signal localization for the exact same sample changed from junctional to nuclear after going back to it an hour later (1st and 17th images below). All samples are fixed in 4% PFA and not permeabilized to prevent disruption of cholesterol.
Here are summary Max IP images:
My usual protocol is as follows:
- Fix in 4% PFA for 10-15 minutes.
- Wash in Tris-Glycine for 10 minutes (2X).
- Remove Filipin stock (abcam) from -80 C and dilute 1:100 in either PBS (or buffer provided by abcam).
- Incubate in the dark 30 - 60 minutes.
- Wash in PBS for 5 minutes (2X).
- Image same day.
Everything was kept in the dark and DAPI-free throughout the process. To be sure the signal is not unique to the two-photon laser, I checked the signal afterwards with our epifluorescent microscope and observed the same loss of signal/nuclear labeling.
One concern we’ve considered is the laser intensity is too high and is cooking my samples, leading to degradation of Filipin and autofluorescence of the nuclei. While it’s possible there was uneven labeling with the Filipin between coverslips, that doesn’t explain why the same coverslip showed completely different localization after only 1 hour, despite fixation.
Any feedback is appreciated!