I applied immunofluorescence on primary cultures of breast cancer cells and on cells from the established cell line MDA-MB-231 for the localization of cytokeratin 8, using the primary monoclonal antibody human anti-cytokeratin 8, purchased from Sigma (code No: C5301). My problem is that the immunofluorescence protocol that I followed, gave perfect results in the breast cancer cells of the culture cell line, while in the cells of the primary culture it didn't work (the immunofluorescence was very weak and obscure).
The cells are permeabilized with Triton X-100 0.5 % diluted in PBS for 5min. Could the cells of the primary culture need different permeabilization time compared with those of the cell line?
Could someone help??
Thank you in advance
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