Hi,

I have not used AO in vivo but the problem with AO is that it is a DNA intercalating staining. It is not specific for apoptosis but will stain all DNA. In vitro you look for fragmented nuclei to judge apoptosis. Maybe for in vivo it is more sensible to use a different staining such as TUNEL or Annexin V, which are specific for apoptosis.

Best of luck with your experiment.

Hello, Theventhiran.

I agree with Joanna, AO is not a specific dye for apoptosis and have to be used in complex with another methods or dyes.

But there are some points of view that AO can be used as vital pH indicator in the living cell (pH transition from alkalescent to weak-acid is manifested in changing of AO spectrum from green area to red). Since the cytoplasm acidulation is a relatively reliable apoptotic hallmark, you can use AO in this way. Make an overlay image with your FITC- and red filters and compare green/red ratio in the areas of interest.

See the similar approach in attachment.

Lucky staining!

Hi Joanna,

Thank you so much for the advice. I was considering to switch to TUNEL assay and now I'll definitely do that. Cheers :)

Hi Dmytro,

thank you so much for the suggestion. I'll do that and if I cant get a convincing result, I'll try TUNEL assay instead. Thank you :)

As an added concern, we've noticed quite a bit of background fluorescence when using tricaine as an anesthesia with larvae. You may want to use cold water or eugenol instead.

Hi,

For convincing (and publishable) results, I would use TUNEL or caspace-3 staining for the reasons mentioned above. But I've used AO staining for a rough cell death assay in zebrafish and it did seem to preferentially label dying or dead cells. The patterns I saw were similar to what I saw by TUNEL staining, though not as specific. (It's possible these cells are just easier for the AO to get into, or the fluorescence becomes brighter in acidic lysosomal vesicles?) You should see bright, small, punctate staining amidst some low level background staining. From what I can see, your staining looks more like non-specific, high level background. (The otolith staining an example of non specific). I haven't done this in a long time, but I believe I used a lower concentration, possibly 0.5mg/ml, and washed it out for at least a half hour before imaging. So maybe try a range of dilutions, see what works.

For live imaging, methyl cellulose ( 2-3%) if you can get some, then make a well out of tape, clay, surf wax etc...

Good luck!

Please look at the attached paper. I hope this attached paper maybe of some help.

TUNEL or Hoechst if you have nuclei in your sample/zebra fish.

Hi,

Thank you everyone for your replies. I just tried both Mr.Keith's and Mr.Dmytro's suggestions and pulled out some microscopy images. I did both FITC and red fluorescence imaging and merged them.

You can also confirm the apoptosis using BrdU incubation.

below link may give you some idea.

http://zfin.org/cgi-bin/webdriver?MIval=aa-fxallfigures.apg&OID=ZDB-PUB-070504-3

Hi Theventhiran,

I'm surprised in your AO staining that you don't see staining in the nose or lateral line. When I did AO staining during my PhD, these were always brightly stained. Here's the protocol I used which is basically straight out of the neurodegeneration paper in the 1996 zebrafish issue of Development.

Acridine Orange Wholemount protocol

Make a 1000X stock solution (store at 4C) of 5 mg/mL AO in water.

Add 3 microL stock solution to dechorionated larvae in E3 in a very small dish

(3 mL vol). Swirl to mix.

Let larvae swim in the dilute AO in dark for 15-30 min. I often just put

a towel over them to create "dark".

Transfer larvae to large dish (20 mL volume) of E3 and let rinse for

about 30 seconds. Try to transfer as little of the AO solution into the

rinse as possible.

Visualize immediately under a GFP or FITC filter.

AO fluoresces green, background is orange. Image quickly.

You should see the nose and lateral line light up. These tissues are not

dying, they're just permeable to the AO.

It's easier to see if the fish are also treated with PTU, so that they are

not pigmented. The larvae in the attached PDF have been treated with PTU and then stained with AO.

Acridine Orange staining is nice for a quick and dirty test of cell death, but TUNEL is the gold standard and still easy to do whole-mount or on sections.

Hi Ann,

Thank you very much for your reply. I think I made my mistake in AO preparation. I will try this method and let you know this week. :)