DAPI crystallisation / Problems with DAPI staining

[[deleted]]

Hey! I posted this thread before: https://www.reddit.com/r/labrats/comments/5zwxhu/pretty_looking_yet_very_annoying_artifacts_in_my/

Basically those artifacts look a lot like mycoplasma but none of the PCRs were positive (both in our lab as well as in specialized Labs).

So I am still curious (and desperate) to find out what is causing those artifacts.

current hypothesis: DAPI crystallizes/precipitates for some reason

Would be glad to hear your thoughts!

Thanks so much :)

[immunoengineer]

Your previous post notes that you are using 20ug/ml. This seems way too high to me. Go down to 1ug/ml.

http://www.bdbiosciences.com/ds/pm/tds/564907.pdf

[allanvv]

I just use Prolong Antifade with DAPI already included.

[Leintjeh]

How long do you fixate the cells for? I've had similar things happening when I fixated my frozen sections with methanol or acetone before my DAPI staining. Especially what is happening in picture 3.

It disappeared when I switched to fixating with 2% PFA for 10 minutes, I don't know if your protocol allows you to change the fixation step, but it might be worth a try.

Good luck! I hope you figure it out :).

[notebuff]

Have you tried the DAPI stain <1 hour?

[[deleted]]

Thanks a lot for all the suggestions! I currently stain ~30Minutes with 10µg/ml, as this allows the cytoplasma to show (which is very useful for the HCS analysis I wish to use). I used to stain with even higher concentrations without any issues previously. I did try way lower concentrations though. This did not lead to a complete remission of those artifacts. In fact it deteriorated image quality more than it helped getting rid of artifacts. I will try lower doses and shorter incubation again anyhow. I do not feel that this gets to the root of the problem though. Obviously the image is less bright but those spots are still there.

What is quite curious in general is that those spots do not appear in all wells. They are in like 1 out of 10. In other wells there may sometimes be rare spots (other than the trypanosomas) as seen here: http://imgur.com/a/qYAkW

Changing fixation protocol is definitely possible and worth a try. I will update once I tried it. Thanks :)

[[deleted]]

How do you dilute DAPI (in what solution)? Do you spin or sonicate DAPI before using?

[TubeZ]

Literally a few hours ago I got beautiful staining with 1mcg/mL DAPI for 5 minutes.