Optimized Antibody Labeling and DetectionAntibodies conjugated to fluorescent dyes are well-established tools for examining specific proteins in cells, a method referred to as immunocytochemistry (ICC). This procedure is commonly accomplished either by using fluorophore-conjugated primary antibodies raised against a specific protein, or by first labeling with primary antibodies followed by secondary antibody detection. Optimal labeling and detection methods are critical for achieving high signal-to-noise ratios with immunolabeled cells. For the most robust results, conditions for fixation, permeabilization, and blocking should be optimized in conjunction with careful reagent selection for each cell model and antibody used in your experiments.
Fix. Permeabilize. Block.
Two basic types of chemical fixatives are used in ICC: organic solvents and cross-linking reagents. Because solvent-based fixatives such as methanol and acetone delipidate membranes and dehydrate samples, cross-linking fixatives may be preferable for preserving cell structure in monolayer cultures. Fixation with the cross-linker glutaraldehyde often results in increased autofluorescence, and therefore formaldehyde is generally recommended over glutaraldehyde as a standard cell fixative.
With cross-linking fixatives, a post-fixation membrane-permeabilization step using detergent is required to ensure that large antibodies can enter the cell and bind intracellular targets. Although many detergents have been reported for ICC, Triton X-100 solutions of 0.02–0.1% are typically very effective at permeabilizing cell membranes in fixed monolayer cultures. When using solvent-based fixatives, the cell membrane is permeabilized by the solvent, and thus additional detergent treatment is unnecessary. Optimal fixation and permeabilization conditions may vary depending on antibody access to specific epitopes and should therefore be optimized for each primary antibody.
A blocking step should be performed to reduce fluorescence due to nonspecific antibody binding. A common blocking step is the addition of a 2–10% solution of bovine serum albumin (BSA). Another approach employs the addition of a 5–10% solution of serum from the species in which the secondary antibodies were raised. For example, when using goat anti–mouse IgG secondary antibodies, samples may be effectively blocked with 10% normal goat serum. To further reduce background fluorescence, the Image-iT FX Signal Enhancer can be included in the blocking step to decrease nonspecific fluorophore labeling due to electrostatic interactions between the dyes and the cell constituents.
Labeling and Detection
Relative antigen abundance is an important factor in choosing the best ICC labeling and detection approach for a given model (Figure 1). Low signal-to-noise ratios obtained even after optimizing the fixation-permeabilization-blocking procedure can be caused by a low-abundance target or a suboptimal detection strategy.
For a high-abundance target such as β-tubulin, ICC using directly conjugated primary antibodies provides a bright and specific signal with a simple workflow (Figure 1A). Technologies available for direct labeling of primary antibodies include Zenon and APEX Labeling Kits (Figure 2), which are particularly useful for conjugating small amounts of antibody and for experiments involving multiple primary antibodies from the same host.
For lower-abundance targets, primary detection is also attainable, albeit with more demands placed on both the antibody and the imaging system. By combining a optimally labeled, high-affinity primary antibody with longer excitation light exposures that increase signal integration times, primary detection is achievable on less abundant targets.
For increased sensitivity, primary antibody labeling can be followed by detection with a fluorophore-conjugated secondary antibody (Figures 1B and 3). Our extensive selection of secondary antibodies includes bright, photostable Alexa Fluor conjugates—the reagents of choice for immunofluorescence microscopy. 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 ~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 (Figure 1C) or a streptavidin bridge followed by a biotinylated reporter such as Qdot 655 biotin. Although processing times increase with additional incubation and endogenous biotin–blocking steps, detection sensitivity also improves as a result of the multiply 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 (Figures 1D and 4).
For improved detection sensitivity with rapidly bleaching dyes, our SlowFade Gold and ProLong Gold antifade reagents have been shown to increase photostability and reduce initial fluorescence quenching in fixed cells, fixed tissues, and cell-free preparations.
Figure 1. Four typical labeling and detection approaches for ICC should be considered. (A) Fluorophore directly conjugated to a primary antibody; (B) Primary antibody followed by detection with a fluorophore-conjugated secondary antibody; (C) Primary antibody followed by detection with a biotinylated secondary antibody in conjunction with a fluorescent streptavidin conjugate; (D) Enzyme amplification (e.g., tyramide signal amplification, TSA) using a primary antibody, HRP-conjugated secondary antibody, and fluorophore-labeled tyramide. With secondary antibody and enzyme amplification methods, significant gains in signal-to-noise ratios may be achieved.
Figure 2. Direct antibody labeling. Formaldehyde-fixed and Triton X-100–permeabilized bovine pulmonary artery endothelial (BPAE) cells were blocked with goat serum and stained with DAPI (blue) and Alexa Fluor 488 phalloidin (green) to label nuclei and F-actin, respectively. Tubulin was detected with anti–β-tubulin mouse IgG prelabeled with either (left) the Zenon Alexa Fluor 594 Mouse IgG1 Labeling Kit (red) or (right) the APEX Alexa Fluor 594 Antibody Labeling Kit (red).
Figure 3. Detection with a fluorophore-conjugated secondary antibody. Formaldehyde-fixed and Triton X-100–permeabilized BPAE cells were blocked with goat serum and stained with DAPI (blue) and Alexa Fluor 488 phalloidin (green) to label nuclei and F-actin, respectively. Anti–golgin-97 mouse IgG was detected with Alexa Fluor 594 goat anti–mouse IgG antibody to visualize the Golgi apparatus (red).
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