I am seeing high background outside of my neuronal cells when using membrane potential indicators. What can I do to reduce background?
If you use our FluoVolt Membrane Potential Kit (Cat. No. F10488), the kit provides a background suppressor to reduce this problem. For other indicators, consider the use of BackDrop Background Suppressor (Cat no. R37603, B10511, and B10512).
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Why do I lose all signal from my neuronal tracer when I do a methanol fixation on my cells?
If the tracer you chose is a lipophilic dye and fix with methanol, the lipids are lost with the methanol. If you have to use methanol fixation then choose a tracer that will covalently bind to proteins in the neurons.
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I stained my cells with a lipophilic cyanine dye, like DiI, but the signal was lost when I tried to follow up with antibody labeling. Why?
Since these dyes insert into lipid membranes, any disruption of the membranes leads to loss of the dye. This includes permeabilization with detergents like Triton X-100 or organic solvents like methanol. Permeabilization is necessary for intracellular antibody labeling, leading to loss of the dye. Instead, a reactive dye such as CFDA SE should be used to allow for covalent attachment to cellular components, thus providing for better retention upon fixation and permeabilization.
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I labeled my neurons with DiI and then fixed and permeabilized and now I have no signal. What did I do wrong?
DiI is a lipophilic dye that resides mostly in lipids in the cell, when cells are permeabilized with detergent or fixed using alcohol this strips away the lipid and the dye. If permeabilization is required CM-DiI can be used because this binds covalently to proteins in the membrane; some signal is lost upon fixation/permeabilization, but enough signal should be retained to make detection possible.
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What is the difference between fast and slow-response membrane potential probes?
Molecules that change their structure in response to the surrounding electric field can function as fast-response probes for the detection of transient (millisecond) potential changes. Slow-response dyes function by entering depolarized cells and binding to proteins or membranes. Increased depolarization results in additional dye influx and an increase in fluorescence, while hyperpolarization is indicated by a decrease in fluorescence. Fast-response probes are commonly used to image electrical activity from intact heart tissues or measure membrane potential changes in response to pharmacological stimuli. Slow-responding probes are often used to explore mitochondrial function and cell viability.
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