御返信ありがとうございます。
大変ありがたいですし、参考になるのですが、実績のある方法でないと気軽には進める気にはなりません。
下記の論文は1例ですが、同じGeltrexを使用したものです。
例えばこれを見るとGeltrexが溶けているような印象を受けるのですが、違うのでしょうか。
pmc.ncbi.nlm.nih.gov/articles/PMC8844721/
Fixation:
a.To isolate organoids, first wash with PBS and add 1 mL of ice-cold PBS to each well. Incubate the plate on ice for 1 h, breaking apart the Geltrex dome halfway through by pipetting.
b.After 1 h repeat mixing by pipetting, the Geltrex should now be completely dissolved.
c.Coat a pre-chilled 15 mL sterile tube with 1% (wt/vol) ice cold PBS-BSA (Bovine Serum Albumin). Also coat pipette tips with the same solution by pipetting up and down 3 times before handling organoids.
Inline graphicCRITICAL: Until organoids are fully fixed (step i) coat tubes and tips with 1% (wt/vol) ice cold PBS-BSA before contact with organoids to prevent organoids from adhering to plastic surfaces.
d.Transfer the organoid suspension to pre-chilled 15 mL sterile tubes on ice, combining organoids under the same conditions.
e.Collect any remaining organoids in the wells with 1 mL 1% (wt/vol) ice cold PBS-BSA.
f.Fill up to 10 mL with ice cold PBS, mixing by inversion to ensure the Geltrex has been completely dissolved.
g.Centrifuge organoids at 100 g for 3 min at 4°C.
Inline graphicCRITICAL: Organoids should form a pellet without any remaining Geltrex. Undissolved Geltrex can stick organoids together and impair immunofluorescence by preventing penetration of antibodies or producing high background staining (See problem 3).
h.Organoids are then resuspended in 1 mL ice cold 4% (wt/vol) PBS-PFA (Paraformaldehyde) and incubated on ice for 45 min to fix, mixing the organoids halfway through by pipetting. |
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