Hello everybody,
in our lab we have been struggling with the staining of our organoids. As described in several publications, we do cryosections and stain them, but unfortunately without success.
Does somebody have a good protocol for staining retinal organoids and would be willing to share it?
that would help us a lot!
I would recommand a so called Nissl's stain. You can try it with cresy violet.
The protocol you will find in the attachment. When you are using unfixed , native sections, put them after they dried out into 4% Paraformaldehyd or Formalin over night. Next day rins the sectins short in destilled water, 10 dipps in 70% EtOH, 3 min in 100% EtOH, 3 min in 70% EtOH and the continue as it is discribed in the protocol. Good luck!
Hello there Kevin,
Can you specify the type of staining or otherwise your goal of staining the organoid? The counterstain that Ute Neubacher is likely helpful. But if you were looking for specific targets, as revealed via immunohistochemistry, stain penetration is a hurdle I've seen discussed in the literature. I'll put this here in case it is helpful for you or others; it is an article about doing a whole-mount immunohistochemical stain on corneal tissue.
Article A novel protocol of whole mount electro-immunofluorescence staining
Kevin R. Urstadt You're right, my quetion was a bit vague indeed. We are trying to perform immunocytochemical stainings of specific markers, as a way to characterize our organoids. We do that on cryosections of said organoids.
Ah, I didn't read your initial post so carefully in regards to the sectioning. My apologies, and I hope someone has more precise answers about your ICC. You might want to provide your current protocol/procedure for other readers in case they spot something needing a change.
The cresyl violett stain is helpful to get an overview about the quality of your sections and fixation and for orientation. The immunohistochemistry depends of your target proteins you like to investigate. You can do a cell typisation with GFAP for Glia and NeuN for neurons for example.
Ute Neubacher Thanks for the tips, it might as well be interesting to try the Nissl's stain
Hello Kevin! You might want to check this publication :) Article Hydrogel-based milliwell arrays for standardized and scalabl...
In it, we describe a method to generate and immunostain retinal organoids in arrayed round-bottom hydrogel milliwells (Gri3D plates). The gel array containing the organoids can be embedded in OCT and frozen to be cryosectioned. In the paper, we performed immunocytochemistry for multiple markers, such as PAX6, OTX2, GNAT1-2...
The plates are commercially available at https://www.sunbioscience.ch/. If you have any questions, feel free to reach out, cheers!
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