Hi, I suggest you try TSA kit from perkin elmer, and there is some protocol to make the kit using chemicals, much less expensive but equally efficient. The method can greatly enhance the real signals (you only need to use 1:2000 to 1:4000 dilution of primary antibody)

Thanks for the info. I'll definitely consider this. So you've tried this kit and get good signals. Am I right? 

First of all, how do you know that there should be phospho-Akt?  I would first try some less challenging antibody (total Akt), just to make sure that you can get a good signal. Quite often problem can be with permeabilization.

Hi Danijela, 

Thanks for your reply. I usually use pAkt ser 473 to do the whole mount ICC. I can get pretty good signals. Some people in my lab had tried using pAkt thr 308 to do ICC. The 308 signal is always not as good as 473. You mention something about permeabilization. I checked a lot of online protocols. Some protocols suggest using methanol or ethanol instead of 4% PFA for fixation and acetone for further permeabilization. Do you think this will help? Are there any other options that I can try/ 

Thank you!!

see this:

http://docs.abcam.com/pdf/protocols/Whole_mount_zebrafish.pdf

Also there might be the case that actually there is nice phospho- at Ser473 but not at Thr 308. I know that it might sound contra-intuitive, but as I recall that might me happening in some cancer.

Hope this helps

Hi Danijela, 

I really appreciate your help. I'll try this protocol. 

Thank you!