Hello,
I am working on 3D culture of MDCK cysts. Whenever i try to fix the cells with PFA, the matrix depolymerizes. We thought it was something to do with the matrigel, so we tested different lots as well as the Cultrex matrix, but it was the same case for all. We are pretty confused how all these papers report fixing cysts with 4% PFA, in our hands the matrix dissolves even at 0.2% PFA concentration!
We are able to culture cysts well, but have no idea how to fix and stain them further. Methanol fixation won't work since it dehydrates the cysts and cause them to collapse. Formalin fixation didn't help either. Please advise!
That's strange because we have the opposite issue with Matrigel when using for 3D co-culture of organoids. What concentration of Matrigel are you using? Perhaps you need to increase the concentration in order for the Matrigel to hold it's shape during fixation. Could be a temperature issue also since it will dissolve above room temperature. Try fixing it in cold PFA and see if there is any improvement.
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