Hi all,
I'm currently trying to optimize a protocol for plastic embedding and sectioning human embryoid bodies (EBs) from H9 stem cells. Since I am trying to show that our labs way of developing embryoid bodies is potentially more organized in shape and growth compared to other previous methods for making EBs, I wish to show the 3 germ layer formation, ideally through ICC before embedding and sectioning.
I am looking at this kit from R&D http://www.rndsystems.com/Products/sc022/
but I know there are some varying levels of success with antibodies and plastic embedding...
Has anyone previously used the antibodies that come with this kit, or the kit itself with plastic embedding? If so, what were the downfalls? Better success with ICC before or after embedding?
Basically, any info on these antibodies with use in plastic embedding would be much appreciated!
Hi, I have no experience with this kit you mentioned, nor with embryoid bodies, but I worked hard with immunofluorescence in plastic resin. Basically you have to worry about two things: fixing time and penetration of antibodies. Some penetrate the resin and there are no problem, other must be done before embedding. Some cases of very sensitive antigens to fixing, we have worked with lyophilization, but loses a lot of aesthetic quality of the material. But I can guarantee that controlling fixation time, concentration of antibodies and if necessary some kind of antigen retrieval, you will success with any material. Good luck.
Hi all, thank-you for your answers. There's a bit more confidence added to how I will go about this.
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