Troubleshooting Tips for Fluorescence Staining

Problem: No Staining or Low Signal

Primary antibody is not validated for application

• Check the supplier information to find out if your primary antibody is recommended for your application. If possible, validate the antibody using a positive control cell line or tissue that expresses the target.
• Confirm that the species reactivity of the antibody is compatible with your sample.
• Check that the target protein is expressed in your cells or tissue of interest by searching the scientific literature or The Human Protein Atlas.

Antibody concentration is too low

• Perform a titration of antibody concentration to find the optimal concentration. The optimal concentration for primary antibodies can vary widely; concentrations for initial testing usually start around 1 ug/mL or higher.
• Secondary antibodies are typically used at 1 ug/mL for cell staining and as low as ?50 ug/mL for near-infrared western detection.

Intracellular target is not accessible for surface staining for flow cytometry

• Check whether the protein of interest is expected to localize intracellularly or on the cell surface.
• Even if the protein of interest is localized to the cell surface, your antibody may bind a cytoplasmic domain inside the cell. Check whether the antibody immunogen or epitope is in an extracellular domain of the target protein, or is validated for cell surface staining.
• If the binding site is not known, perform intracellular staining to determine if target is localized inside the cell.

Secondary antibody is not compatible with serum proteins used for blocking

• If using anti-goat or anti-bovine secondary antibodies, avoid blocking buffers with milk, goat serum, or BSA.
• Use IgG-free BSA or fish gelatin for blocking to avoid cross-reactivity of secondary antibodies with immunoglobulins in the blocking agent

Fluorescence is photobleaching during microscopy

• Using mounting medium with antifade, such as EverBrite™ Mounting Medium.
• Some fluorescent dyes photobleach more rapidly than others. Blue fluorescent dyes in particular such as CF®350, CF405S® and CF®405M have relatively lower fluorescence and photostability. We recommend choosing photostable dyes like rhodamine-based CF® Dyes for microscopy applications.

Imaging settings are not compatible with dyes

• Check that you are using the correct excitation/emission settings for the dyes.
• Note that far-red conjugates are not visible to the human eye through the microscope, and must be imaged using a CCD camera or confocal imaging system.

Problem: High Background or Non-Specific Staining

Cell or tissue autofluorescence

• Autofluorescence is a major and nearly universal source of background in tissue sections, and also is present in some primary cells and pigmented cell types.
• Include an unstained control to determine the level of autofluorescence in your sample.
• Cellular autofluorescence is high in blue wavelengths, so avoid using blue fluorescent conjugates such as CF®350, CF405S® and CF®405M for low expressing targets.
• Use our TrueBlack® Lipofuscin Autofluorescence Quenchers to quench tissue autofluorescence.
• Amplify your specific signal over background by using indirect immunofluorescence (primary + secondary antibody) or tyramide signal amplification.
• Check out our Tech Tip: Battling Tissue Autofluorescence for more information.

Cross-reactivity of secondary antibody with other antibodies or proteins in sample

• Perform staining controls with secondary antibody alone to determine whether the secondary antibody is binding the sample directly.
• For multiple staining experiments, stain with each primary and secondary combination separately to detect unexpected antibody cross-reactivity.
• For indirect staining (primary + secondary antibody) with multiple primary antibodies, use highly cross-adsorbed secondary antibodies to prevent cross-reactivity.
• When staining rat tissue with anti-mouse antibodies, use anti-mouse cross-adsorbed against rat.
• Staining of mouse tissues with anti-mouse antibodies (known as mouse-on-mouse staining) may require special protocols to block binding of endogenous antibodies in the tissue.
• Highly charged fluorescent dyes, including CF®405S, Alexa Fluor® 647, or Cy®5.5 can contribute to non-specific binding of conjugates. Specialized blocking buffers such as TrueBlack® IF Background Suppressor System or TrueBlack® WB Blocking Buffer can reduce background from charged dyes.

Fluorescence cross-talk between channels

• For multi-color experiments, perform controls with each stain alone, and image the single-stain controls in all channels to determine whether there is fluorescence cross-talk or bleed-through of dye fluorescence between channels. Use the same gain/exposure settings that you would use to detect specific staining in each channel when checking for fluorescence cross-talk from other channels.
• Choose dyes that are spectrally well-separated for multicolor imaging. Our Fluorescence Spectra Viewer can be useful for evaluating dye separation for multicolor experiments.
• Multi-color flow cytometry analysis may require fluorescence compensation. See your cytometer user manual for information.
• Confocal microscopy imaging settings can be optimized to minimize cross-talk by limiting cross-excitation during scanning, or by changing the emission cut-off for different dyes.
• For DAPI bleed-through into the green channel, reduce the concentration of DAPI, or optimize confocal imaging settings to prevent cross-talk. Far-red nuclear counterstains for the Cy®5 channel, such as RedDot™2, also can be used to avoid this problem. Check out our Tech Tip: Avoiding Artifacts from UV Photoconversion of DAPI and Hoechst for more information on troubleshooting staining issues with DAPI and Hoechst blue nuclear dyes.

Blotting membrane autofluorescence

• Autofluorescence can vary widely between different types and sources of blotting membranes. Scan an unused blotting membrane next to your western blot to detect membrane autofluorescence.
• In our experience, nitrocellulose and low fluorescence PVDF membranes show similar background fluorescence, but PVDF can give higher sensitivity, possibly due to higher protein binding.

Suboptimal western blot blocking

• Test different blocking agents to find the optimal conditions, or try a blocking buffer specifically designed for fluorescent westerns, like the TrueBlack® WB Blocking Buffer Kit.

Insufficient washing of western blots

• Increasing the number of washes can improve background for western blots. Use a generous volume of wash buffer with rocking so blots move freely during washing.

Antibody concentration too high

• If both signal and background are high, antibody concentration may be too high.
• Perform a titration of antibody concentration to find the optimal concentration. The optimal concentration for primary antibodies can vary widely; concentrations for initial testing usually start around 1 ug/mL or higher.
• Secondary antibodies are typically used at 1 ug/mL for cell staining and as low as ?50 ng/mL for near-infrared western detection.