Dear Researchers,
I want to perform a whole mount ISH experiment on the zebrafish ovary. There are many papers in which they have done this experiment but the details are kind of missing. What I usually get to read is 'XYZ protocol, mostly Thisse et.al. protocol is followed'. I tried it twice but somehow did not work so well. My probe appears excellent on the gel. Another thing is the amount and the duration of the proteinase k treatment. Once I tried without proteinase K and the second time with proteinase K, 5ug/ml for 15 mins. Since there is no activation of the egg, the chorion is not hardened. Moreover, I am not sure if Proteinase K works on the chorion. Actually, in both the cases, early stage oocytes are stained quickly. The problem is with the late stage oocytes which are quite big.
Please let me know if there is any modification in the standard protocol.
Thanks a lot.
It's been ages since I've worked with Zebrafish so I'm not certain of the dimensions of your tissue. Years ago (1990s) working on mouse embryos, we encountered issues with ProK of erratic batch to batch variation - this could cause terrible problems with morphology disruption or generation of background because you could never be certain of how potent the protease was from one week to the next without extensive testing - which was not easy when the tissue supply was limited. An alternate protocol that was less prone to over-digestion of the tissue was to use the classic detergent buffer RIPA at the equivalent stage for permeabilisation, incubated at 4˚C for varying durations depending on the exact embryonic size. This proved so successful in my hands that I have never used ProK since, and whenever a colleague has encountered problems with ProK I've suggested this as an option, mostly with positive results.
There are many versions of RIPA if you look online. The variant I used to use is something akin to the following :
50mM Tris pH7.4-7.5 (although pH8.0 comes to mind here)
1% NP40 (also known as Nonidet, IgePal CA-630 etc)
0.5% Sodium deoxycholate
0.1% SDS
150mM NaCl
As I don't have my thesis to hand, I can't remember off the top of my head if our version had EDTA in it, but this can be present up to 1-2mM - I need to look this up when I have time and will update here if necessary.
Good luck.
Dear Dr. Dickinson,
Thanks a lot.
I was completely unaware of this alternative for ProtK. I will give it a try. I have a recipe for RIPA buffer as we use it regularly for WB. Actually, the problem is the chorion since we dissect out the complete ovary and then tease out different stages of oocytes, so, we can't use pronase or any other mechanical method to remove the chorion as it is in the close proximity of the egg. So, I was looking for some alternative technique or at least want to know if the ProtK treatment works on the oocytes we chorion.
I will try your method and see if it works.
Thanks a lot again.
As your ovaries and chorions etc are going to be quite small, you may find that the RIPA step will be quite short. From memory we would switch from PBS into RIPA for small numbers of minutes - 2-3 changes of 2 / 3 / 5 / 10 minutes, then back to PBS for 2-3 changes depending on exact embryonic stage of mouse embryos, but no more than this. You may even need to use less than the above.
Be aware also that most RIPA recipes usually have inhibitors of various types present for their normal cell / WB applications, and some of these may actually impact on ISH stages - EDTA on the magnesium needed for alkaline phosphatase activity, phosphatase inhibitors also could affect AP activity etc, so you might need to omit some components of your house recipe if these are present. The important parts are the buffer conditions, the salt, and the detergents.
best of luck!
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