I need to look for the expression of certain proteins in organoids which are embedded in Matrigel. Is it possible to fix organoids in collagen or Matrigel and later stain the organoids for markers?
Hi Shilpa Patil
I have been working with brain organoids, which are embedded in Matrigel at an initial step of the protocol and thereafter are grown in agitation. In this case, it works just fine fixing it in PFA 4% in suspension, including in OCT, and then proceed to cryosection for downstream procedures such as immunostainings.
I am not sure if that is your case or if you need a protocol for fixing while in Matrigel. Anyway, find attached our latest work (still not peer-reviewed) with the full protocol described. I hope it helps!
https://www.biorxiv.org/content/10.1101/2021.06.16.448740v1
Hello Shilpa Patil
Growing organoids embedded in solid-ECM can be tricky. Moreover, the subsequent immunofluorescence protocols are usually lengthy, and require organoid release with the risk of losing the microtissues, and/or modifying their original morphology.
I would suggest that you try Gri3D for your organoid cultures. https://sunbioscience.ch/products/ With these microwell plates, you can grow organoids of any type in little (or no) presence of ECM, in suspension-like conditions. You can do your immunofluorescence protocol on the plate and image without having to do any transfer steps!
Check out how Gri3D works in the following publication:
Article High-throughput automated organoid culture via stem-cell agg...
Feel free to contact me for more info, cheers!
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