Hello to all,
I would like to do Immunofluorescent Staining experiments on organoids. So far, I don't know how to do it properly...
First, I tried to do it "in suspension" (after removal the matrigel, i pelleted the organoids and did the IF in a 15 mL falcon), but i lost more than the half of the organoids.
So, i thought maybe, after removing the matrigel, to plate the organoids on coated Coverslips before fixing them (to avoid the lose). Has anyone ever did it ?
Merci !
Hello Chaima
You can have a look at the research article below. May be it might help you.
Article Generation of Pancreatic Ductal Organoids and Whole‐Mount Im...
Regards.
most people treat the organoids as small pieces of tissue and use frozen immunohistochemistry, check out some of the papers from my team Article Comparison of Developmental Dynamics in Human Fetal Retina a...
or https://pubmed.ncbi.nlm.nih.gov/26283078/
You get beautiful results if ytou do this. why not paraffin? because the antibodies work better on frozen, because it's hard to pass such small pieces of tissue through clearing etc. solutions and then find them, because it works great in frozen PFa-fixed organoids. All you have to do is fix, wash, embed in OCT, freeze in small molds and then section using a cryostat, and mount sections on + charged slides. Then treat as normal IHC slides. If you want to stain the whole large organoid then you need to permeabilize them, triton x-100 is a good reagent. Then clearing them is probably the only way to see through the whole organoid. I doubt this is a reliable solution. Sectioning and then 3D- reconstructing the organoids using the optical z sections is probably a better approach. Though tedious. Wholemount may work but it is hard to focus when taking the images
Thanks for all your replies !
So, for now, I did the whole mounting, and it worked really well (thickness about 30µm; staining ChromograninA in green). I manage to have beautiful pictures. I did the staining "in suspension".
I'm wondering now if it's possible to remove the organoids from the matrigel and pour them on a pre-coated slides with chambers.
Hi Chaima,
We are using Smear Gell to mount and fix the organoids with very good results. I hope the contribution will serve you.
Best.
https://www.diagnocine.com/Product/Smear-Gell/52446
Hi Chaima,
I fix intestinal organoids directly on coverslips, following this protocol
Article Immunofluorescent Staining of Mouse Intestinal Stem Cells
Hope it helps
Thank you all for your replies ! I'll give a try and let you know asap !
Dear Chaïma Ayachi ,
Immunofluorescence on organoids embedded in solid-ECM can indeed be tricky! I'm glad you managed to optimize your protocol.
If you'd like to further streamline the procedure, 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. A pipetting port on the side allows media/buffer exchange without disrupting the organoids. You can do your immunofluorescence protocol directly on the plate (from fixation with PFA to antibody incubation), 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...
If you have any questions, feel free to contact me, cheers!
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