We are developing monoculture spheroid model.
Spheroids are developed in Ultra low attachment round bottom well plate and transferred to OCT cryomold and cryosectioned. Cryosections are of 5 microns. These sections are treated further as follows:
1) PBS wash to remove remnant OCT using dropper
2) Fixation in 4% PFA
3) PBS wash using dropper and Tap water wash
4) Hematoxylin staining for 3-5 minutes
5) Tap water wash
6) 1% acid alcohol treatment- Dipped twice,
7) Wash with tap water
8) Bluing in saturated lithium carbonate- Dipped twice
9) Wash with tap water
10) Dipped in 95% alcohol
11) Eosin treatment- 20-30 seconds
12) 95% alcohol and absolute alcohol- 2 times each, xylene two times
13) mounting in DPX
However, we observed the attached image. We are not able to figure out what has gone wrong. Kindly help us in this regard.
Whether the issue lies in sectioning or staining?
Hi, as we work with tumor spheroids, we also tried to perform stainings for the spheres, using either cryo- or parrafin sections. We actually found that paraffin embedding works far better, as it helps to preserve the sphere structure, wheras cryo-sectioning usually results in a worse outcome. You can see two HE stainings as an example attached. If you wanna perform the cryo-sectioning nevertheless, i'd advice you to perform PFA fixation before embedding.It should definitely give better results than the one shown in your image.
Best
Firstly I would ask how did you process your spheroid for cryosectioning?
What I would do is a pre-fixation treatment with 4% PFA in PBS for 30 minutes followed by an overnight incubation in 12% sucrose as a cryoprotectant. Then I would embed your spheroid in a solution of 7.5% gelatin/12% sucrose and allow to solidify at RT. Then I would cut out a block of the gelatin, with your spheroids as closely as possible to the edges of the block. I would then place the gelatin block in a cryo-mold and re-embed in blue OTC (so you can see the block better) and flash freeze the whole thing in an isopentane bath with dry ice (~ -60 degrees C). Store the mold in -80oC until ready to section. Place your sample on a cryostat with the stage set at 1oC and the chamber at -20oC and cut sections of 15-20 um (even 30 if you can). Immediately mount on slides and proceed with your staining protocol. Re-fix your sections with PFA once on the slide. Good luck!
Hi dear Manish,
Simply, just fix the spheroids with formalin for 24 hours and it is better to fix multiple spheroids in the same try (about 20 or more). Then in the next day spin them and embed them in wax and normal sectioning with about 14um or less depend on the size of your spheroids. After that keep them on the glass slide and stain them in regular method.
Feel free for any further qeustions, please
Best wishes
Sarmad
Thank you very much for the suggestions. Will implement the same. Sarmad Al-Sahaf Dario Pacitti Sascha Seidel
Sascha Seidel Is it possible to do tunel assay(to visualize the apoptotic nuclei) in paraffin emmbeded spheroids?
I personally never tried a TUNEL staining in those embedded spheroids, usually do cleaved Caspase-3. I don't see a reason why a TUNEL staining shouldn't work, though.
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