Our protein shows an apparently different localisation pattern between cultured cells and animal tissues. IF-staining of HeLa, SHSY5Y and primary skin fibroblasts are both nucleolar and cytosolic with the same distribution for GFP-tagged protein in living HeLa. However, IF and IHC with paraffin embedded mouse tissue shows only cytosolic and no nucleolar localisation.
The antibody recognizes both human and mouse. Same for a homologous isoform using a different antibody: If nucleolar and cytosolic in cultured cells, but only cytosolic in mice tissue. No idea what the protein's function actually is, but it binds RNA and it can be isolated from the nucleus in complex with splicing factors, so nucleolar localisation would not sound artificial. In cell culture, the protein also seems to exit the nucleus upon generic stress (heat, oxidation, intoxication), which suggests some sort of shuttling properties. Any idea of what might have happened? Methodical artifact? Metabolic issues of cultured cells?
What are the differences in fixation and processing of cells and tissue? Which pretreatment (antigen-retrieval) do you perform in IHC? Is the antibody able to stain in the antigen in formalin-fixed tissue?
My first approach would be to enhance the antigen-retrieval in order to "release" also the nuclear antigen and to increase the sensitivity. Maybe cellular antigen is abundent enough for visualisation and nuclear antigen is not. (?) just my thoughts.
Hi Fabio, I was working with NPXY receptors and there is a huge difference in their localization in tissues compared to cell lines.
I could never find any explanation for this, but it might be due to the radically different cell environments.
Additionally, cell lines are selected for their ability to grow as a monoculture and often what they express and how they express it can't be compared to their in vivo counterparts.
Hi Fabio,
In cell culture, cells are grown submerged forming a monolayer. Also, submerged cultures are generally prepared with a single cell type. In any tissue, cells are organized in a three-dimensional environment and receive different input/stimuli from surrounding cells, other cell types and extracellular matrix. This could be the reason why you are observing different localization in cell culture vs. animal tissue.
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