Immunofluorescence Microscopy

There are usually three aspects to dye stability: 1) chemical stability of the dye core structure; 2) stability of the reactive group; and 3) photostability of the dye.

Chemical stability of the dye core structure:

This refers to resistance of the dye core structure to decomposition caused by factors other than photo-bleaching. These factors may include temperature, pH and incompatibility with other chemicals in the medium. This type of stability information is most useful for estimating the shelf-life of the dye that is already covalently attached to another molecule (e.g., an antibody), or for assessing the chemical compatibility of the dye in certain applications. CF® Dyes bear the core structures of coumarin, pyrene, rhodamine or cyanine dyes, all of which are known to have excellent chemical stability. In general, CF® Dyes are far more stable than the antibodies they label. Thus, if a CF®-labeled antibody loses activity during storage, the problem is not likely to be caused by the dye. CF® Dyes are also stable enough for labeled nucleic acids to be used in PCR or nucleic acid hybridization, where high temperature is involved.

Stability of the reactive group:

Reactive CF® Dyes comprise a reactive group used in bioconjugation. Among the various reactive groups, only amine-reactive succinimidyl ester (SE) and thiol-reactive maleimide groups are unstable because the small amount of moisture trapped in or leaked into the packaging vials can cause hydrolysis of the reactive groups over time. The SE group, in particular, is susceptible to degradation. Thus, in order to slow degradation, CF® Dyes comprising these reactive groups must be stored at -20°C under anhydrous conditions. Furthermore, stock solutions of the dyes must be made using dry solvents, such as anhydrous DMSO. One advantage of CF® Dye SE products over other commercial dyes is their relatively high stability. Normally, an SE group can be derived from either an aliphatic or an aromatic carboxylic acid group, but an aliphatic carboxylic group tends to result in a more stable SE, offering higher resistance to hydrolysis and thus better labeling efficiency. All of the CF® SE Dyes have their SE groups derived from aliphatic carboxylic acid groups, unlike many commercially available SE dyes that are often prepared from aromatic carboxylic acid groups.

Photostability:

This refers to the dye’s ability to withstand photobleaching. For most dyes, photostability is not a major problem for routine handling under ambient light or for applications, such as flow cytometry and Western blotting, where the dyes are only briefly exposed to light. However, for microscopy, especially for confocal microscopy, where the dyes may be subject to intense illumination for an extended period of time, photobleaching can be a major concern. Similar to the photostability of other fluorescent dyes, both the dye core structure and the structure-modifying groups attached to it play a role in the photostability of CF® Dyes. CF® Dyes bear the core structure of rhodamine, cyanine, pyrene or coumarin dyes; among the four types of core structures, rhodamine core is the most photostable, followed by cyanine and then by pyrene and coumarin cores. The structure-modifying groups and the way they are attached to the dye cores are a key innovative aspect of CF® Dye technologies that contributes to the superior photostability of CF® Dyes over that of other commercial dyes. In general, rhodamine-based CF® Dyes, whose wavelengths range from visible to the near-IR region, offer the best photostability, making the dye ideal for microscopy applications.

Yes, EverBrite™ mounting media are compatible with a wide range of dyes. Unlike VectaShield®, EverBrite™ won’t interfere with cyanine-based fluorophores like Alexa Fluor® 647, Cy®3, or Cy®5.

AlexaFluor is a registered trademark of Thermo Fisher Scientific; Cy Dye is a registered trademark of Cytiva; VectaShield is a registered trademark of Vector Laboratories.

EverBrite™ wet-set mounting medium is an aqueous, glycerol-containing medium. It can be removed from samples by washing several times with PBS or other buffer. However, washing with PBS will not remove DAPI staining.

For coverslips mounted on slides with EverBrite™ Hardset or EverBrite™ wet-set medium, the slides can be soaked in PBS in a slide jar for several hours with gentle agitation until the coverslip slides off (avoid prying or pulling the coverslip, which could damage the sample). Then wash the slide several times to remove any remaining mounting medium. If the coverslips have been sealed with CoverGrip™ Coverslip Sealant, soaking in PBS will remove the sealant as well. Other methods may be required to remove other types of coverslip sealants.