Hello everyone!
When I search the protocol of whole mount in situ hybridization, I found the time often from E8.5 to E11.5 or E12.5.
Whether the time point of whole mount in situ hybridization cannot exceed E12.5.
For example E18.5 cannot do whole mount in situ and it can only do single tissue in situ?
Any suggestion will be appreciated!
It depends on what you are looking for. The mouse embryo develops so quickly that you may want to target a particular time point (to the hour) for a specific protein or gene. Best of luck.
Of course it depends on your scientific question, but I think that you would get much more information performing in situ on sections. According to my experience, after e12.5, the embryos are becoming bigger and less transparent, so not only it is more difficult to stain the deep tissues, it is also difficult to image, and to understand. On the contrary on sections, you would be able to identify the positive and negative tissues very easily. You also may try to section embryos after you have performed the whole mount.
Thanks for all of your help!
And to Audrey, would you meaning that I have to section the whole embryo old than E12.5?
I don't do ISH before, but I wonder whether the embryo is bigger for section and maybe it is difficulty to fix the embryo.
Yes I think that at e12.5 already it is worth sectioning the embryo, because you would get access to much more information: you could recognize the developping organs, tissues, and even cell types, so you would get a much more precise expression pattern.
And in addition, I think that all the steps : permeabilization, incubation with probes or antibodies, would be more efficient and better controlled on sections. For the fixation I have performed ON incubation at 4 degres in 4% PFA for e16.5 and it was ok.
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