We are working on staining the specific proteins to check for its localization. We also working of live/dead staining assay simultaneously. Since the cells are embeded in the hydrogel, we could not get better staining even after trying different methods.
Kindly help if anybody got better technique to proceed with.
Hi
This reference could help you
A gel-free 3D microfluidic cell culture system
What kind of hydrogel are you using for your 3D cell culture? As long as the material does not interact with the stain you often can use standard protocols for 2D cell culture but with increased incubation times.
Dear Christopher Aronsson ,
We are using GelMA hydrogel.
yes, that's the major issue; even the gel is catching the stain and it's creating lot of background and could not image the cells properly.!
Any tips to avoid this??
I usually use Alexa Fluor 488 - Phalloidin to stain F-actin and DAPI for nuclei. You may include additional fluorescent markers for you proteins too. Then, by confocal microscopy, you can obtain great images from z-stacks and/or build your own 3D reconstructed images/videos.
You would need fixation to perform this assay, just in case this means an inconvenience...
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