Hello everyone,
i'm trying to generate 3d spheres of mesenchymal stem cells. when i had to perform ICC, trasferred my spheres into a plastic dish , i let them settle down till they attached. i performed the steps of fiaxation and permeabilization and everything was ok , i didn't lose any of them.
after the overrnight incubation in blocking solution, i checked my spheres before going further. i noticed that almost all my spheres collapsed only 10% remained.
i'm doubting about my fixation step but i'm not sure since my method worked several times.
Have you an idea about this issue? i'm planning to try a coated coverslip to enhance the adherence, or an embedding gel to maintain the structure ( like collagen). in the perfect case oct would be the best but i'm at the first steps of characterization and i need whole-mount images. Also i heard about chambered slides but is it any good at this step ?
Yassine Messat there are two possibilities for this problem.
1. MSC's are plastic adherent, hence if allow MSC''s spheroids to attach to the plate the cells might disintegrate from the spheroid and your spheroids will collapse.
2. Avoid vigorous shaking or rocking of your plates while staining because if the spheroids are not compact enough they might collapse.
Using non-adherent plates is advisable.
you can embed in matrigel, if you want to avoid presence of any factors inducing the spheres (e.g., TGFb, BMP, EGF, etc., present in matrigel) you can embed in hydrogel, there are many available. As previous person replied, the spheres attach, but then many detach quickly (since you give them only o/n time to attach), and leave a footprint (circle of cells) behind. But a bigger question is -why to stain this way, you will not stain the core of a sphere. We routinely used to section retinal organoids (also spheres, blobs of stem cell-derived retinal tissue) using cryostat, after fixing w PFA and embedding them in OCT (optimal cutting temperature) solution and freezing. We treat spheres as mini-pieces of tissue. You can then do IHC on sections, and even reconstruct sphere in 3D using stacks of IF images from confocal, using metamorph or volocity, e.g.,
PMID: 26283078, or/and other papers we published. If you really determined to do ICC only, stain the spheres in 96-well or 48-well plate or in an epp tube (i.e., don't let them attach, stain in solution). People stain histological sections this way, too ("floating sections"). Then add a lot of viscous antifade medium, coverslip and image. You can even avoid adding a coverslip if spheres are large, but add antifade. Good luck
You can ocpy crotamine, abasic polypeptide, will attach and help endocytosis, garanteed
If you develop your method, it's possible. can you tell me what was your protocol and what kind of serum did you use?
Igor O. Nasonkin can you please recommend me a protocol about floating staining . i would be grateful
PMID: 19609935, this is a classical approach for staining PFA-fixed sections of brain and spinal cord, used by many neuroscientists. The huge advantage is that you dont need to mount all sections,
https://www.mskcc.org/sites/default/files/node/3816/documents/free-floating-immunohistochemistry.pdf , google "free floating sections. You keep them in the mixture of ethylene glycol (antifreeze), glycerol and 10x-PBS (diluted to 1x with water: i believe 30% Eg, 30% glycerol, 10% 10x PBS and water 30%, google, its freely availabe. Great method. you use brush to pick selected sections, rinse 2-3 times 10-15 seconds each in 1x PBS to get rid of cryoprotectant, then block 45 min-1h (overnight ok), then do primaries, then wash and do secondaries as for usual IHC. You mount on the slides after 2ndary stain and nuclear counterstain are done. If the sections are thicker than ~35 microns, then the center is not stained well (due to AB penetration from both sides). When you mount and dehydrate (yes, you do it for IF-labeled cryosections too), sections shrink (e.g., 30 u section becomes ~12 micron thick), which helps confocal.
i will be more than happy to guide you further, this is a superb method, routine by those who work in neuroscience but an eye opener for those, who has not been exposed.
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