Firstly, is 30-min proteinase K digestion enough? Have you tried any probe as positive control under the same experimental conditions?

Thanks Jorge...I had used PTU from just before 24 hpf. Should that be ok or earlier would be better?

Whole mount ISH at 5dpf is a challenge; perhaps you should consider doing ISH on sections? Penetration of the probe will be the main problem in my opinion; pigmentation also as PTU treatment will be 100% efficient only shortly after treatment and cannot be applied at later stages (ineffective in most cases)

What part of the embryo is your probe trying to access? In my experience the trunk is particularly impermeable at 5 dpf, so you may have to ProK longer, or consider sectioning before ISH.

PTU from just before 24 hpf should be fine, as long as you don't see any pigmented melanophores, but this won't affect probe penetration.

From what I've noticaed, the success depends mostly on the probe. Some of them just are too weak. And indeed, a positive control is a good idea.

I would definitely go first for ISH on sections, or cut the embryo into parts, e.g. head on one side, body on the other side.

I suggest using 10µg/ml ProK and treatment for 1.5 hrs. Fix ProK treated embryos with 4%PFA for at least an 1 hour. Also use a probe that you know it works as a control.

Adding PTU before 24 hpf is fine. Melanization actually starts around 28 hpf. You don't want to add PTU prior to completion of gastrulation as this will cause abnormalities. After fixation, when you dehydrate your embryos in methanol, be sure to have them in methanol at -20 deg C for at least overnight. As for the protK incubation, 30 mins is ok. And I agree with Gokhan above, fix your embryos in 4% PFA after protK. Definitely try out a positive control probe first. In my experience the tips of the tails tend to be a little "sticky" and you may find a few embryos with their tails sticking to one another. Be gentle when you try to disconnect them.

Hi Gnaneshwar. Although I haven't done any Zebrafish insitus, I have done some Hydra whole mount RNA insitu. I have too faced some such problems and had to do lot of attempts to standardize the procedure before finally getting good results. In my opinion, when you attempt whole mount insitu quality of your probes matter a lot along with the right ProK treatment. ProK treatment is not only specific to what specimen you are using, but also how long a probe you use. Longer probes requires longer or more stringent ProK treatment. But you should standardize this since you dont want to ruin your sample by treating it so much that the tissue is damaged to an extent that you have non-specific staining. I would suggest you use smaller probes (ranging between 200-500bp long). I have myself successfully used probes as long as 1.2Kb but I dont think it will possible for your case. I think you should probably focus on these two aspects.

3 X 1hr water washes post fixation can improve penetration in the trunk, but under fixed embryos will turn into "soup". Re-using your probe improves signal and decreases background. Use a known, strong probe to get your protocol working. Using diluted coloration buffer, at 4C, and changed every couple days, can bring up week signal without blowing out your background. At 5dpf sections and then In Situ, are a lot of work for minimal gain. I'd get a good proven probe, run through a series changing fixation times and ProK treatments, do a matrix, see what works best. On your new probe, use it on some throw away embryos (might as well see if it comes up but just to get a sense of the development time) and then use it on your perfected protocol. Section post In Situ, if you are looking at deep structures.

We use 10ug/ml Proteinase K to treat the 5 days old embryos for 30 min and can get good results. Remember to refix the embryos in 4% PFA for 2 hrs. The quality of the probe is most important and it is usually used at 1-2 ug/ml with 600-800bp length. Do it with a positive control is a good idea.

I suggest looking at this paper

Zebrafish embryonic neurons transport messenger RNA to axons and growth cones in vivo.

Baraban M, Anselme I, Schneider-Maunoury S, Giudicelli F.

J Neurosci. 2013 Oct 2;33(40):15726-34. doi: 10.1523/JNEUROSCI.1510-13.2013.

Best

Hi Ganesh,

You should definitely follow Tor's suggestions. Using a strong probe to troubleshoot your protocol problems is a good idea.

We PK 5 dpf embryos for up to 2 hrs using PK at 10ug/ml. Then post-fix for 20 minutes.

You should also try longer hybridizations, troublesome probes often work much better with 48 or 72 hr hybs (e.g. over the weekend). Longer hybridization is so much better that we do it always.

Ptu conditions have been optimized to balance the potential lethality and mutagenic effects of early treatment against the importance of getting them started before pigmentation begins at 24 hpf (not 28). The results are published in Karlsson, Hofsten, and Olsson, Marine Biotechnology 2001. Briefly, begin treating with 75 uM ptu at 22 hpf for best results.

For shrinkage, make sure you always re-hydrate (go from MeOH to aqueous) in 5 min steps, 25% H2O, 50% H2O, 75% H2O, 100%.

Hi, talk to your fishes. Hyb time you say for 5 dpf maybe is too short, do not try to shorten the protocol, too much prk treatment increases background, wash in MABT for at least 2 days, develop signal in glass, nbt bcip proportion should be 1 to 10.

Check this paper and good luck.

mab21l2 transgenics reveal novel expression patterns of mab21l1 and mab21l2, and conserved promoter regulation without sequence conservation

In my case, I have used proteinase K at 20 ug/ml concentration for 15 min. More than 20 min, your fish 5dpf would be sticky. 

Hi Nguyen Thanh Vu did you try 15 min PK (20ug/ml) for 15 min for 5pdf zebrafish ? coz pk digestions vary from stage to stage and also the concentration one uses matters. I ask this because, for 72 hpf or 3 dpf i tried 20 min pK (20ug/ml) of concentration, which means for 5 dpf it must me longer ?