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Dyes that carry multiple negative charges can introduce background. Usually, this is more of a concern with labeled antibodies that carry many dyes, as opposed to a small toxin like bungarotoxin. When staining tissues, the endogenous autofluorescence of the tissue itself is often the most significant source of background. Endogenous fluorescence background in tissue is usually highest in the blue wavelengths (DAPI channel) and lowest in the far-red (Cy®5 channel). Our CF®633 bungarotoxin (catalog no. 00009) is a far-red conjugate for the Cy®5 channel with a low negative charge that should have low background from either the dye or autofluorescence.

We test fluorescent bungarotoxin on rat skeletal muscle sections. While the tissue shows autofluorescence, the bungarotoxin staining of motor endplates is usually much brighter than the background for all of the dye colors we’ve tested. However, if you are staining human tissue (especially brain), lipofuscin autofluorescence may be bright in all channels. This usually shows up as bright, punctate dots around cell nuclei. While we would usually recommend our TrueBlack® lipofuscin quenchers for human brain tissue, they are not compatible with bungarotoxin staining. We have, however, found that EverBrite TrueBlack® Mounting Medium (cat. no. 23017) can be used to mount skeletal muscle sections stained with bungarotoxin.

Cy Dye is a registered trademark of Cytiva.

We do not have firsthand experience with LPS labeling, but according to the literature, LPS has been labeled using amine-reactive dyes, like FITC. Our amine-reactive CF® Dye Succinimidyl Esters should also work for this. There is a publication for enzymatic labeling of LPS using dye hydrazides. Our CF® Dye hydrazides could be used in this method. The paper also describes the traditional amine labeling method and purification of the conjugate.

If you order an oligo to be synthesized with an amino group on one end, then our CF® Dye succinimidyl ester (SE) dyes could be used to label it using a standard amine labeling protocol for DNA.

We recommend using an HPLC purified oligo, with an amino group with a C6 linker at the 5′ or 3′ end. To remove free dye after labeling, you could use G25 Sephadex, gel purification, ethanol precipitation, or HPLC, depending on the degree of purity needed.

Alternatively, this may be something that an oligo synthesis company may be able to perform for you as a custom labeling.

Reactive dyes for bioorthoganol conjugation (click chemistry) including alkyne, azide, BCN, TCO, and methyltetrazine can be used to prepare polymers. We do not offer CF dye methacrylate at this time, please contact techsupport@biotium.com to inquire about custom synthesis.

We don’t have direct experience or customer feedback on CF dyes for 6-color imaging on LSM 800, but because this instrument has tunable excitation and linear unmixing, you should have a lot of flexibility in dye choice. We would also recommend contacting Zeiss support to see if they have advice for selecting dyes for multi-color experiments for the instrument.

The violet excited 405/430 dyes are generally less bright and should be used for more abundant targets. The 754 dye would also be recommended for more abundant targets. Even with optimal excitation, the GaAsp detectors on the Zeiss LSM 880 are expected to be less responsive to NIR wavelengths. The other dyes are very bright and would be better for less abundant targets to minimize cross-talk.

Below we’ve listed the dyes we’d usually recommend for the standard laser lines on the LSM 880.

405 nm laser line

CF®405S, Ex/Em 404/431 nm; recommended for abundant targets

458 nm laser line

CF®430, Ex/Em 426/498 nm; recommended for abundant targets

488 nm laser line

CF®488A, Ex/Em 490/515 nm; bright and photostable

Note: CF®488A and CF®514 must be separated by spectral imaging if used together

514 nm laser line

CF®514, Ex/Em 516/548 nm; dye for spectral imaging

Note: CF®488A and CF®514 must be separated by spectral imaging if used together

561 nm laser line

CF®568 Ex/Em 562/583 nm; very bright and photostable

Note: CF®568 and CF®594 must be separated by spectral imaging if used together

594 nm laser line

CF®594, Ex/Em: 593/614 nm; very bright and photostable

Note: CF®568 and CF®594 must be separated by spectral imaging if used together

633 nm laser line

CF®640R, Ex/Em 642/662 nm; CF®640 will be brighter with on-center excitation

CF®660R, Ex/Em 663/682 nm; CF®660R is off-center for this laser, but very bright and extremely photostable; less likely to have cross-talk with CF®594

Tunable 2P Laser 720-1020

CF®754, Ex/Em 745/786; recommended for abundant targets (LSM800 detectors are expected to be less sensitive for NIR dyes)

Biotium developed TyraMax™ Amplification Dyes as high-performance TSA dyes, offering brighter, more photostable signals than Aluora® and Opal® reagents. They also remain stable in amplification buffer for up to 24 hours, facilitating automated staining workflows. TyraMax™ Dyes are offered as standalone dye solutions, as 3-color or 5-color dye sets plus DAPI counterstain, and in a sampler for custom panel optimization. See below for alternatives to specific Aluora® and Opal® reagents. Learn more about our reagents for tyramide signal amplification (TSA).

TyraMax™ Amplification Dyes

TyraMax™ 3-Plex Amplification Dye Set with DAPI (Cat. No. 33029)

TyraMax™ 5-Plex Amplification Dye Set with DAPI (Cat. No. 33030)

TyraMax™ Amplification Dye Spectral Sampler (Cat. No. 33031)

Aluora is a registered trademark of Thermo Fisher Scientific; Opal is a registered trademark of Akoya Biosciences.