Qdot™ Streptavidin Sampler Kit

Here are our recommendations:

Use one of our extensive selection of secondary antibodies conjugated to bright, photostable Alexa Fluor dyes. The degree of labeling for each conjugate is 2-8 fluorophores per IgG molecule, with potentially three secondary antibody-binding sites per primary antibody, providing signal amplification of approximately 10-20 fluorophores per primary antibody.
Alternatively, primary antibody labeling can be detected with a biotinylated secondary antibody in conjunction with either a fluorescent streptavidin or a streptavidin bridge followed by a biotinylated reporter such as Qdot biotin. Although processing times increase with additional incubation and endogenous biotin-blocking steps, detection sensitivity also improves as a result of the labeled streptavidin.
For low-abundance targets, signal amplification may be necessary for optimal signal-to-noise ratios. Tyramide signal amplification (TSA) is an enzyme-mediated detection method that utilizes the catalytic activity of horseradish peroxidase (HRP) to generate reactive fluorophore-labeled tyramide radicals. These short-lived tyramide radicals covalently couple to nearby residues, producing an amplified fluorescent signal localized at the HRP-target interaction site.
For improved detection sensitivity with rapidly bleaching dyes, our SlowFade Diamond or ProLong Diamond antifade reagents have been shown to increase photostability and reduce initial fluorescence quenching in fixed cells, fixed tissues, and cell-free preparations.
Please review this web page for further optimization tips (https://www.thermofisher.com/us/en/home/references/newsletters-and-journals/bioprobes-journal-of-cell-biology-applications/bioprobes-issues-2011/bioprobes-66-october-2011/guide-to-immunocytochemistry.html).

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A common method for amplifying antibody detection is biotin-streptavidin detection, where a biotinylated secondary antibody is combined with subsequent labeling with a dye-conjugated streptavidin. This will amplify the signal by approximately 2-8 times, but endogenous biotin must be blocked beforehand. Another option is to use tyramide-signal amplification, where a horseradish peroxidase conjugate is used with a dye-labeled tyramide. This will amplify the signal by approximately 10-20 times, but endogenous peroxidase will need to be blocked. A final option may be to use a Qdot nanoparticle antibody or streptavidin conjugate, which can yield a signal as much as 40 times higher than a standard organic dye conjugate, depending on the Qdot color.

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Here are some suggestions: Use the Qdot Incubation Buffer (Cat. No. Q20001MP). The included buffer is formulated specifically for improved signal-to-background ratios in most immunolabeling applications using the Qdot streptavidin conjugates. Alternate buffers may result in more variable staining and, in particular, may increase background staining. However, some specific applications may require other buffer conditions. Please see the protocol "Double-labeling Using Qdot Streptavidin conjugates."
Determine if the sample has a high level of endogenous biotin. Block the sample using an avidin-biotin pre-blocking step.
If you have used the Qdot Incubation Buffer and still get high nonspecific background, then it may be necessary to check other steps of your procedure. Blocking the sample with BSA or normal animal serum will generally decrease nonspecific binding of both antibodies and Qdot streptavidin conjugates. It is a good practice to dilute your primary and secondary antibodies in the blocking buffer. Some tissues such as spleen and kidney sections may contain endogenous biotin, which may contribute to non-specific signal. Endogenous biotin can be blocked with an avidin/biotin blocking kit (Cat. No. E21390).
Grainy staining or clumps of fluorescent material appear in the background.
Occasionally the BSA within the Qdot Incubation Buffer shows slight aggregation over time. It is necessary to remove this aggregate prior to labeling the sample with the Qdot streptavidin conjugate. Spin down the incubation mixture before addition to the sample. This can be accomplished by spinning the samples in a benchtop centrifuge (Eppendorf 5415) at 5,000 x g for 2 minutes. The material can also be passed over a 0.2 µm spin filter unit before you add it to the sample for staining to remove microscopic precipitates. If you are using a buffer that is different than the Qdot Incubation Buffer, this behavior can often be attributed to higher levels of NaCl or other salts in the incubation buffer, and may not be easily fixed with filtration. In this case, reduce the overall salt concentration.
Optimize concentration of biotinylated secondary antibodies.
Optimizing specific signal can often be achieved by adjusting the level of biotinylated antibody used instaining. High levels of biotinylated antibody are necessary to obtain specific labeling, but overly high levels will contribute to nonspecific binding of the antibody to the sample. Nonspecifically bound biotinylated antibody will bind to the Qdot streptavidin conjugate, resulting in higher staining of the background.
Optimize concentration of Qdot streptavidin conjugate.
Just as titration of primary and secondary antibodies is necessary to achieve optimal specific signal in immunolabeling applications, the level of the final probe should be optimized for each conjugate. In general, concentrations at or slightly below saturation should have the optimal signal-to-background ratio, while concentrations substantially higher than saturation will compromise the assay with higher background levels.

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Here are some suggestions:

Confirm imaging/detection setup suitability.
Make sure that you are using an appropriate filter set to detect the signal. Please consult Table 1 in the Qdot Biotin User Manual for a list of appropriate and optimal filters.
Check to see that Qdot conjugate is fluorescing using an alternative light source.
Qdot conjugates will normally fluoresce brightly under a hand-held ultraviolet lamp (long wave, such as the type used to visualize ethidium bromide on agarose gels). Although we have not seen pronounced loss of fluorescence of these materials under any storage conditions that we have investigated, we have not been able to examine all storage conditions. If the Qdot product does not appear to fluoresce under the long wave UV excitation, please contact Technical Support at techsupport@qdots.com. For a microscope, perform a spot test: place a small droplet (2 to 5 µL) of the quantum dot solution onto a clean slide (no coverslip) and examine under the appropriate filter set at low magnification.
Confirm the specificity and titer of primary antibody.
Make sure the antibody will recognize the intended targets. Make sure there is sufficient primary antibody bound to the targets. This verification can be performed by ELISA-based capture of the antigen of interest, or by other techniques that can be found in lab manuals such as the Current Protocols in Immunology.
For Qdot streptavidin conjugates, confirm biotinylation of antibody.
Make sure your antibodies are effectively biotinylated. It may be necessary to independently adjust the concentration of both the primary and secondary antibodies used in the assay to obtain optimal signal and minimal background.
PAP pen ink may quench signal.
Use an alternate method for isolating target areas on the slide. If your protocol requires the use of a PAP pen, we recommend the ImmEdge Hydrophobic Barrier Pen (Cat. No. H-4000) from Vector Labs.

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Spinning your ITK Qdot nanocrystals at approximately 3,000 rpm for 3-5 minutes should remove the white precipitate from the supernatant. Use the supernatant immediately.

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