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Organelle & Cytoskeleton Stains

Cationic dyes are widely used as mitochondrial probes. They accumulate within the cell and preferentially localize in the mitochondrial matrix, induced by the greater negative membrane potential of mitochondria in live cells compared to the plasma membrane potential. Membrane potential plays a direct role in governing the distribution of the dyes across the plasma membrane: the more negative the potential, the greater the accumulation of the positively charged dyes. MitoView™ 633 is a cationic lipophilic dye that is potential-dependant and accumulates in mitochondria in proportion to the electron gradient similar to classic dyes like TMRM, TMRE and Rhodamine-123.

Ratiometric dyes like JC-1 constitute another class of potential-dependant mitochondrial dyes. In normal healthy cells, JC‐1 accumulates in the mitochondria and undergoes aggregation in a potential‐dependent manner, whereas in unhealthy, apoptotic or dying cells with dissipated mitochondrial potential, the dye delocalizes to the cytosol where it dissociates to the monomeric state. The aggregated dyes (J-aggregates) fluoresce red, while the monomeric dyes fluoresce green.

Some mitochondrial‐specific dyes are considered “structural” dyes, as opposed to “functional” dyes, because they are capable of staining mitochondria regardless of their polarization status. The fluorescence of cells stained with these dyes is directly proportional to their mitochondrial content or “mitochondrial mass.” MitoView™ Green is a potential-independent dye that accumulates in the mitochondria and interacts with the mitochondrial matrix via hydrophobic interactions. It can be used to stain mitochondria in live as well as formaldehyde -fixed cells, but fixed cell staining tends to be less specific compared to live cell staining. MitoView™ Green staining is also not compatible with solvent-based fixatives or permeabilization. For fixed cell staining, we recommend using one our CF® Dye conjugated mitochondrial marker antibodies. Nonyl Acridine Orange (NAO) is another potential-independent mitochondrial dye that binds specifically to cardiolipin in the inner mitochondrial membrane.

Mounting medium can alter the staining of lipophilic dyes like LipidSpot™, due to interaction of the dyes with glycerol or other components that help form the interface between the coverslip and slide. The antifade compounds in mounting medium are generally compatible with the dyes. In our tests, LipidSpot™ staining was well preserved in EverBrite™ Mounting Medium (catalog. nos. 23001/23002) for up to 24 hours after mounting, but lipid droplet size and staining intensity were somewhat altered after samples were stored in mounting medium for several days. Therefore, if mounting medium is required to image samples, we’d recommend imaging as soon as possible after mounting.

LipidSpot™ is not compatible with FluoroShield mounting medium (staining is lost immediately after mounting). We have not tested other types of mounting medium.

Most of our MitoView™ dyes are sensitive to mitochondrial membrane potential, and therefore can be used in live cells only.

MitoView™ Green is relatively insensitive to mitochondrial membrane potential, and can be used to stain formaldehyde-fixed cells (for best results, we recommend first fixing, then staining). MitoView™ Green does not stain well with samples that are fixed with methanol, permeabilized with detergent, or paraffin embedded. For best results staining mitochondria in fixed cells or tissue sections, we recommend using one our Mitochondrial Marker Antibodies, available with a wide selection of bright and photostable CF® dyes and other conjugations.

ViaFluor® Live Cell Microtubule Stains are cell-permeable taxol probes for imaging the microtubule cytoskeleton in live mammalian cells. We do not have permeability data for these probes to the plant cell wall. Based on literature, plant tubulin binds taxol weakly as compared to animal tubulin, so staining may be weak or non-existent. We do offer low-cost trial sizes for evaluation.

Intracellular lipid droplets are cytoplasmic organelles involved in the storage and regulation of lipids. The LipidSpot™ Lipid Droplet Stains are fluorogenic neutral lipid stains that rapidly accumulate in lipid droplets. They become brightly fluorescent in the presence of neutral lipids like triglycerides and cholesterol esters.

LipidSpot™ stains may be used on fresh or PFA-fixed tissue cryosections, similar to Nile Red or BODIPY dyes. However, lipid droplets may not be well fixed in tissue sections, so discrete lipid droplet staining may not be preserved. Paraffin embedded sections are not recommended, because paraffin tissue processing extracts cellular lipids. Similarly, methanol or acetone fixation of cryosections may extract lipids, and is not recommended.

Mounting medium also may disrupt LipidSpot™ staining. We’d recommend either mounting tissue sections in PBS, or using a glycerol-based mounting medium like EverBrite™ Mounting Medium, and imaging within one day of mounting. FluoroShield Mounting Medium is not compatible with LipidSpot™ staining.

TrueBlack® and TrueBlack® Plus are hydrophobic in nature and quench lipofuscin autofluorescence mainly through hydrophobic interactions. They could therefore stain/bind to lipid structures and quench the fluorescence signal of BODIPY and other lipid droplet stains. Also, original TrueBlack® #23007 is used in 70% EtOH, which could interfere with lipid droplet morphology and affect staining. TrueBlack® Plus #23014, on the other hand, can be used in buffer instead of 70% ethanol, and may be more suitable for combining with lipid droplet staining. However, since the binding of TrueBlack® Plus is also dependent on hydrophobic interactions, we recommend testing TrueBlack® pre-treatment and post-treatment for compatibility with lipid droplet staining in your sample type.

LysoView™ dyes are fluorogenic dyes that contain weakly basic amines and accumulate in the low pH environment of lysosomes as well as other acidic compartments in the cell including early and late endosomes. Although the fluorescence of LysoView™ 540 and LysoView™ 640 is sensitive to increasing pH, it would be hard to specifically distinguish early from late endosomes or lysosomes based on florescence intensity. Using antibodies against early or late endosomal markers or compartment-specific  (GFP or other) fusion constructs would be more appropriate.

Other options that may be suitable for monitoring endocytosis and vesicle trafficking include the CF® Dye Dextrans, CF® Dye Hydrazides, CF® Dye Human Transferrin Conjugates, CF® Dye Cholera Toxin Subunit B, and Nerve Terminal Staining Kits.

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