I'm trying to detect intracellular iron levels in my cells in-vitro and stained using prussian blue. I fixed the cells in 4% paraformaldehyde, washed and let them sit in prussian blue mix from abcam for 30 minutes before washing and mounting them. When I checked the cells were stained a brownish color instead of blue. Has this happened to anyone before? Does anyone have any suggestions or know what's going on?
To get a blue Prussian Blue pigment there has to be Fe3+ in your cells. Iron tends to be washed out in tissue during fixation in aequous solutions. The HCl in the Prussian Blue-mixture liberates Fe3+ out of compounds like Ferritin. These have to be captured by the Hexacyanoferrat to produce the blue pigment. In tissue-staining the insoluble pigment is captured within the tissue-architecture. I don't know, if minute amounts of Iron in a cell can be detected in this way.
I assume, that the brown staining is just an uptake of Hexacyanoferrat to your cells.
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