I'm looking for a quantifiable way to visualize cell-cell contacts in a 3D spheroid. So far, my stainings have somehow worked, but not to my fullest satisfaction (blurry, uneven staining of core etc). I used several antibodies (synapsin, PSD95, actin) or stains (Phalloidin, DAPI), all of them work very well in 2D cultures. Would you recommend to clear the spheroids before staining? Any recommendations for protocols?
Are your spheroids non-adherent? If so, I have previously stained suspended stem cell spherical aggregates by placing them OCT with minimal media or PBS (in a 1.5mL tube cap). I then immediately froze the caps filled with OCT and cells and moved over to the cryosectioner. The OCT should come out of the caps easily. Proceed with cryo-sectioning (10uM) onto a positively charged slide (multiple slices per slide). Briefly fix the samples for ~10 mins and move to a normal ICC staining procedure. Good luck!
Hi Benjamin, I also have blurry, uneven staining of core. Did you find a solution ?
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