I am new to the field of calcium imaging. I used OGB-1 AM dye at 2 uM concentration for 1 hour at 35 C in adult rat hippocampal slices. Only neurons in the stratum radiatum were loaded and the pyramidal cells were not. Bootman et al 2013 claim in their article that if they stain cell cultures above room temperature they inhibit the multidrug resistance-associated proteins (MRP) for better dye loading. Can MRP activity cause also uneven dye loading in the slices? I would be grateful for any suggestions.
Yes it is true if stain cell cultures above room temperature they inhibit the multidrug resistance-associated proteins (MRP) for better dye loading. MRP activity may also cause uneven dye loading in the slices it is also true. Calcium-binding proteins are proteins that participate in calcium cell signalling pathways by binding to Ca2+. The most ubiquitous Ca2+-sensing protein, found in all eukaryotic organisms including yeasts, is calmodulin.Intracellular storage and release of Ca2+ from the sarcoplasmic reticulum is associated with the high-capacity, low-affinity calcium-binding protein calsequestrin. For example, the neuron-specific calexcitin has been found to have an excitatory effect on neurons, and interacts with proteins that control the firing state of neurons, such as the voltage-dependent potassium channel. Compartmentalization of calcium binding proteins has been noted within cells, suggesting that these proteins perform distinct functions in localized calcium signaling. It also indicates that in addition to freely diffusing through the cytoplasm to attain a homogeneous distribution, calcium binding proteins can bound to cellular structures through interactions that are likely important for their function
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