Hi all,
I have troubles with fixation for smFISH method. I use the protocol from the Wormbook. I have tried the freeze cracking (eggs and adults),
then adult fixation without freeze cracking (always 4% paraformaldehyde or formaldehyde in PBS). Even DAPI does not stain the samples,
therefore I conclude that I am facing fixation problem.
Does someone have an idea how to troubleshoot it?
Any experience appreciated,
Thanks in advance for any suggestion.
an obvious question I know, but did you heat-treat, filter and pH your paraformaldehyde solution before using it?
Yes, I did, everything looked correct.
OK. Assuming you are correct and you have a fixation problem (e.g. likely too short to get enough tissue penetration) and that you are using 5ng/ml DAPI, then you could try 100ng-1µg/ml DAPI for 30 minutes @RT (in the dark).
You need to get the DAPI up and running before you have a hope of seeing Ab-labelled fluorescence.
Thank you for the suggestion. I tried longer incubation yet without increasing the DAPI concentration. Because I see a lot of DAPI dye, just not entering the worm but on the surface of the worm. In addition, I am wondering, if DAPI cannot enter (that must penetrate easily), how the probes could penetrate the worm?
we are still with the Fixation Problem then aren’t we?
Yes, indeed, that is why I doubt the higher concentration of DAPI can solve the problem…
Really, there are not that many steps in the Wormbook Methods ‘Fixation of Worms’ smFISH protocol where something could go pear-shaped.
In the absence of smFISH experts flocking to your aid or you contacting Ni Ji / Alexander van Oudenaarden then I have some general suggestions that may get your DAPI working but may not be gentle enough for smFISH hybridisations. I use it more generally.
Resuspend (pelleted, washed worms) in 1 mL fixation solution and transfer to microcentrifuge tube. Keep rotating at room temperature for 45 min.
[Fixation solution: 4% paraformaldehyde (PFA) in 1x PBS]
(you could try a more stringent 8% PFA solution in M9/H2O for 1 hour @RT)
Wash twice with 1 mL 1x PBS.
(wash Worms with PBS until solution is clear)
Resuspend in 1 mL of 70% EtOH. Keep rotating for overnight (or longer) at 4°C. Store at 4°C for up to a month.
(make sure you have removed all the PBS then suspend Worms in 95% EtOH for 1 Minute. If necessary remove EtOH and add fresh aliquot to your worms.
Wash away EtOH with a least three washes with PBS)
Wick off most of the residual PBS (not dry though), mount and add the DAPI (say 10uL of a 10ugmL-1 solution).
See if this improves your strike rate.
Steve
Thank you for your time and suggestions, I will try it.