I have to fix the hepatocytes that has been cultured in 3D environment. i.e. matrigel and collagen. We have cultured the cells in 96-well plates. So after sample introduction, we want to fix the cells and stain them. Could somebody help?
You can fix them the same way as on 2D; i.e. using 4% PFA. These can later be processed in the usual fashion for IF/IHC imaging.
What about the matrigel in which the cells are embedded? How can I free the cells from the matrigel? Or should I fix them in the gel?
Hi Shaima,
if the cells are necapsulated into the matrigel you should increae fixing time (30 to 60min), otherwise if the cells are seaded in the top is enopugh 15min in 2-4% PFA at 4°C.
Good luck
Hi all
After fixation of the cells in matrigel how to stain the cells ? should the matrigel be solubilized to release the cells from the matrigel on to the slide for staining?
If yes does anybody have a protocol for that . Please help.
Dear Betty,
normally after fixation we perform whole moutn IF in the 3D structure, therefore you do not need to solubilize matrigel to relase cells, is enough permeabilize the gel with triton. Otherwise you could make cryosection.
Good luck
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