Hi everyone,
Could anyone who has experience with performing whole gel immunostaining of 3D cell cultures share some tips and troubles they have had when optimizing their protocols. I work with many different sorts of hydrogels and often culture cells in the hydrogel. I reading several protocols intended for immunostaining of the entire gel, I have noticed lower primary antibody concentrations than when compared to the 2D counterpart, and overall longer incubation steps. In a few protocols I have seen the use of permeabilization steps even for the staining of proteins which are cell-surface. Does permeabilization (with Triton X-100, for example) have any effect on the hydrogel itself? Are higher or lower concentrations of blocking solutions typically use?
Hi Sebastian,
we perform routinely 3D construct IF, we use the same antibody concentration as in 2D, the difference are longer and stronger permealization (0.3% triton X-100 in PBS) and longer incubation in primary antiboy (over night), and the seconday antibody we keep for at least 2 hours.
Good luck
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