Immunofluorescence (IF) is a technique where researchers study the location and expression of antigens in cells and tissues using an antibody labeled with fluorescent dye. In IF, secondary antibodies bind to the primary antibody to detect proteins and antigens. The secondary antibody is directly conjugated to or allows for subsequent coupling to a fluorescent molecule (fluorophore) for target visualization under a microscope. This blog covers important considerations for secondary antibody selection, including labeling method, species reactivity, antigen of interest location, optimal fluor conjugates, and signal amplification or multiple labeling (multiplexing) requirements. 

Direct vs. indirect labeling 

An important initial consideration is the labeling methodology and whether to use multiple antibodies (indirect IF) or a single conjugate (direct IF). Direct labeling is a faster method with a single staining step, wherein a primary antibody is conjugated to a fluorophore without the need of a secondary antibody. In indirect labeling, an unlabeled primary antibody is incubated with the sample that binds the target epitope followed by an incubation with a fluorophore-conjugated secondary antibody that binds the primary antibody. Though slower, indirect IF has higher sensitivity, provides signal amplification, and allows for detecting multiple targets on the same sample (1). 

Species considerations  

When performing indirect IF, the secondary antibody should be raised against the host species used to generate the primary antibody. For example, if a primary antibody raised in mouse is selected, the compatible secondary antibody would need to be reactive to mouse, but raised in a different host species (e.g., a rabbit anti-mouse). Furthermore, to prevent the secondary antibody from cross-reacting with endogenous immunoglobulins (IgG) in the tissue, the primary antibody needs to be derived from a different species than that of the sample.  It’s also imperative to consider the isotype of the primary antibody when selecting the secondary antibody. Polyclonal antibodies are generally IgG isotypes and therefore, the secondary is typically anti-IgG heavy and light (H+L) chains to provide coverage for the wide variety of isotypes that make up the population. Monoclonal antibodies often have a specific isotype and subclass, such as IgG1. It’s also been described that IgG subclass-specific secondary antibodies are superior to generic IgG secondary antibodies in several tissue applications in which mouse monoclonal antibodies were used (2). 

Fluorescent label assignment  

Secondary antibodies are commonly conjugated with fluorescent labels, but for greater signal amplification, biotinylated secondary antibodies can be employed alongside a streptavidin-conjugated fluor. Particularly for fluor-conjugated secondary antibodies in multiplexing experiments, consideration needs to be given to the fluor assignment to ensure minimal overlap in spectral emission. Co-localization of the fluors in the sample can often be mistaken instead of bleed through.  For example, Alexa Fluor 488 fluorescence is known to bleed through into the Cy3 detection channel. Scanning the specimen with the individual lasers sequentially and detecting fluorescence in each channel to coincide with laser illumination to produce a more accurate merged image of fluorophore distribution can minimize this problem (3). Another consideration is assigning fluor-conjugated secondary antibodies so that lower expressing targets are tagged with brighter fluors than higher expressing epitopes. For example, if performing an IF panel in mouse spleen with CD3 and CD11c, CD3 would be a more highly expressed antigen because of the density of T cells present in the tissue (21–25% of all cells) while CD11c, which is expressed on dendritic cells, is only expressed on 1–3% of all cells. Thus, in picking fluor-conjugated secondary antibodies, best practice would be to select a dimmer fluor for CD3, but a brighter fluor for CD11c (4). 

Signal amplification 

For each primary antibody, polyclonal secondary antibodies can recognize multiple epitopes to increase binding and signal levels. Similarly, multiple fluorochrome-protein (avidin or streptavidin) complexes can bind to a single biotinylated secondary antibody to increase signal levels. A combination of these methods can result in greater signal amplification. Tyramide signal amplification is another method for enhanced detection. The secondary antibody for this method needs to be conjugated to an enzyme like horseradish peroxidase. The mechanism for this procedure is based on a catalytic reporter deposit in close vicinity to the epitope of interest (5).   

Multiple labeling (Multiplexing) 

Multiple labeling, or multiplexing, helps researchers study the co-expression or co-localization of different targets within cells and tissues. Secondary antibodies are often involved to more effectively probe antigens with higher sensitivity. To achieve multiplexing IF, a standard method is to first incubate with a primary antibody then follow the incubation with the secondary antibody conjugated to the fluorochrome. After that, an additional blocking step is necessary to prevent any trace of the current primary antibody from being recognized by the next secondary antibody. Next, another combination of a primary antibody and secondary antibody can be applied to the tissue, but a fluorochrome that does not overlap with the previous needs to be used to be compatible for imaging.  Researchers studying host-cell and pathogen interactions have demonstrated multiplexing IF with polyclonal and monoclonal antibodies raised in the same species (6). When the primary antibodies are raised in different species, there are several products that combine secondary antibody reagents for multiplexing in a cost-effective, efficient format, such as VectaFluor™ Duet Immunofluorescence Double Labeling Kit, DyLight™ 594 Anti-Rabbit (red), DyLight™ 488 Anti-Mouse (green) . Secondary antibodies are also available in formats that may have improved multiplexing performance. Commonly, secondary antibodies are available as the whole IgG, but they also come in Fab fragments, which are monovalent portions of the antibody that still bind antigens without the Fc structure. These molecules have been demonstrated as effective secondary antibody selections for multiplexing IF studies (7). In particular, they are useful for blocking the surface of IgG’s for double labeling primary antibodies from the same host species (8). 

We hope this blog post gave you valuable information so you have the confidence to perform IF using secondary antibodies. For more resources on IF and secondary antibodies, check out our IF Resource Guide, and stay tuned to the blog for more tips and tricks.  

References 

1. Im K, et al. 2018. An Introduction to Performing Immunofluorescence Staining. Biobanking. Methods in Molecular Biology.  
2. Manning CF, et al. 2012. Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary is of Primary Importance. PLoS One.  
3. Olympus. 2022. Spectral Bleed-Through Artifacts in Confocal Microscopy.  
4. Miltenyi Biotec. 2022. MACS Handbook, Mouse Cells and Organs, Mouse Cell Sources, Spleen (Mouse). 
5. Faget L, et al. 2015. Tyramide Signal Amplification for Immunofluorescent Enhancement. ELISA. Methods in Molecular Biology.  
6. Gachet-Castro C, et al. 2021. Double Labeling Immunofluorescence Using Antibodies From the Same Species to Study Host-Pathogen Interactions. Journal of Visualized Experiments. 
7. Ollington, B, et al. 2021. Immunoresponsive Tissue-Engineered Oral Mucosal Equivalents Containing Macrophages. Tissue Eng Part C Methods. 
8. Brown  JK, et al. 2004. Primary antibody-Fab fragment complexes: a flexible alternative to traditional direct and indirect immunolabeling techniques. J Histochem Cytochem.  

“My staining didn’t work” is a common lament among novice and expert scientists alike performing immunohistochemistry (IHC) and immunofluorescence (IF). A particular nuisance in immunostaining is the appearance of background staining, which is the non-specific staining of tissue and cell elements by colorimetric or fluorescence dyes. Researchers have difficulty identifying the source of the background staining because IHC and IF experiments involve multiple reagents including the tissue fixatives, blocking solutions, primary/secondary antibodies, and detection reagents. In this post we will discuss common sources of background staining and offer tips and tricks for resolving these issues.

This blog post will be the first of a three-part series intended to help researchers overcome different parts of the IHC and IF staining processes that might present challenges, so be sure to stay tuned for other insights that could help your workflow achieve better staining results.

Sample Fixation 

In IHC and/or IF, tissues are fixed with reagents prior to immunostaining and visualization to preserve the tissue and the molecular processes occurring in the specimen. This process helps researchers more easily probe proteins, transcription factors, DNA and/or RNA to study internal cellular processes. During fixation— particularly with aldehyde-based reagents—the hydrophobicity of tissue proteins is increased because a chemical reaction during the fixation process causes cross-linking of reactive epsilon- and alpha-amino acids within and between adjacent tissue proteins (1). This hydrophobicity can contribute to increased background staining in IHC protocols or autofluorescence, which is when biological specimens naturally emit light after absorbing light when visualized under a fluorescent microscope, in IF-based experiments (2). 

The fixation duration, formulation and tissue-to-fixative ratio can impact the degree of nonspecific background staining and autofluorescence (3). For example, the tissue-to-fixative ratio, as well as short fixation times, can cause incomplete fixation with cross-linking only occurring at the edges of the tissue, leaving the center relatively unfixed. This can result in uneven staining, with more intense background staining in the center of the tissue (4). Additionally, over-fixation of a sample can contribute to high tissue autofluorescence (5).  

The researcher should test different fixative reagents, incubation times and tissue-to-fixative ratios in order to determine which is best for their application or search the published literature for methods that are validated with similar tissue types. 

Intrinsic Tissue Features 

Tissues can contain varying levels of extracellular elements like elastin, collagen, red blood cells, and lipofuscin. Like fixation, these structures can contribute to high autofluorescence and obscure specific signal from probes and stained targets.  

Utilizing an autofluorescence quenching reagent, such as Vector^® TrueVIEW^® Autoflurorescence Quenching Kit can reduce background introduced both by tissue components and from aldehyde fixation as discussed above. TrueView is an aqueous solution of a hydrophilic molecule that binds electrostatically to non-lipofuscin sources of autofluorescence like collagen, elastin, and red blood cells, effectively reducing their intrinsic fluorescent signal. To learn more about how TrueVIEW could help overcome autofluorescence in your workflow, check out our blog post, How to improve your immunofluorescence by overcoming autofluorescence. 

Another possible solution for autofluorescence is Sudan Black, a fat-soluble dye with a high affinity for lipids. It can be used for lipofuscin sources of autofluorescence, although the mechanism for this effect is still unknown (6). 

Primary Antibody Titration 

High background can occur because of oversaturation of the target epitope with primary antibody during immunostaining. A thorough titration of antibody alongside a positive control of known target expression is a helpful test to elucidate optimal signal-to-noise in the sample (7). The antibody staining concentration that often yields highly specific staining with the least amount of background can be less concentrated than recommended by manufacturers, so it is important for an individual to determine the best concentration in-house for their application (8). 

Primary Antibody Cross-Reactivity 

Cross reactivity of the primary antibody with other tissue epitopes can cause non-specific staining, therefore an appropriate blocking agent such as BSA (Bovine Serum Albumin), normal serum, or nonfat dry milk is important to ensure proper labeling.  

Species-on-species immunostaining refers to utilizing a primary antibody raised in the same species as the target tissue. If a primary antibody is raised in the same species as the tissue (e.g., a mouse primary antibody and mouse tissue), it is important to use a compatible species-on-species blocking reagent to prevent the generation of a non-specific signal from tissue immunoglobulin (Ig) alongside your primary target. Reagents such as M.O.M.^® (Mouse on Mouse) Blocking Reagent or H.O.H.™ (Human on Human) Immunodetection Kit are suitable in these scenarios. 

Secondary Antibody Cross-Reactivity 

The secondary antibody can also contribute to background staining. The cause is often non-specific binding to endogenous Ig in the tissue—particularly in the previously described species-on-species experiments.  

In the case of a mouse-raised primary antibody used on mouse tissue, the secondary antibody also needs to target mouse (i.e., an anti-mouse Ig), but the secondary antibody can potentially bind to the endogenous Ig in the mouse tissue, causing background staining. Blocking the tissue with an endogenous Ig species-on-species blocking reagent can effectively eliminate any concerns of the secondary antibody binding to the tissue.  

An anti-mouse IgG secondary may also bind Ig in rat tissue due to the similarity in species. To prevent non-specific signal generation in this case, a species-adsorbed secondary antibody, such as a rat-adsorbed anti-mouse IgG from the previous example, can be utilized as an effective option to prevent cross-reactivity (9).  

For suspected cross-reactivity, an alternative is to add 2% or higher concentration of normal serum of the same species as the tissue to the secondary antibody diluent. If that does not work, the secondary antibody concentration can be reduced to minimize background staining.  

Secondary Antibody Deletion Control 

A general method for determining if the source of background staining is due to non-specific binding of the secondary antibody is to test a “deletion control.” To perform deletion control, simply follow the staining protocol for the tissue but omitting the primary antibody. Considerable staining in this control would indicate that the staining is non-specific and potentially originating from the secondary antibody binding to endogenous immunoglobulins in the tissue or to other off-target antigens. That being said, further deletion controls (i.e., where both the primary and secondary antibodies are omitted) are needed to indicate if the background staining is actually coming from the secondary antibody. 

Detection System 

The detection system for IHC often involves an avidin-biotin complex for amplification of the target signal. A deletion control with both the primary and secondary antibodies omitted would demonstrate the contribution of this detection reagent to any background signal. Positive staining in this deletion control would indicate non-specific binding of the detection reagents, and the tissue would most likely need an avidin/biotin blocking step to prevent the reagents from binding to endogenous biotin in the sample. This is particularly apparent in certain tissues that have elevated levels of endogenous biotin like the kidney, liver, and brain (10).  

Substrate 

If IHC background staining occurs after the addition of the substrate alone, then it is possible that endogenous enzyme in the tissue is developing the chromogen. For example, several tissues have considerable amounts of endogenous peroxidase including the liver, spleen, tonsil, lymph nodes, and kidney (11). For horseradish peroxidase (HRP) systems, an appropriate blocking reagent for endogenous enzyme is hydrogen peroxide. For alkaline phosphatase (AP) systems, levamisole can be added to the secondary antibody diluent.  

Some reagents, such as BLOXALL^® Endogenous Blocking Solution, can quench endogenous activity from both alkaline phosphatase and peroxidases, making multiple enzyme detection in one experiment possible. 

We hope the above tips were helpful and get you on the road to better staining. For more tips and tricks to improve your staining, check out our IHC and IF Resources, and stay tuned for more help on the blog.  

References 

1. Ramos-Vara JA. 2005. Technical Aspects of Immunohistochemistry. Veterinary Pathology. 
2. Paavilainen L, et al. 2010. The impact of tissue fixatives on morphology and antibody-based protein profiling in tissues and cells. Journal of Histochemistry and Cytochemistry. 
3. Kim SW, et al. 2016. Immunohistochemistry for Pathologists: Protocols, Pitfalls, and Tips. Journal of Pathology and Translational Medicine. 
4. van Seijen M, et al. 2019. Impact of delayed and prolonged fixation on the evaluation of immunohistochemical staining on lung carcinoma resection specimen. Virchows Archives. 2019. 
5. Baschong W, et al. 2001. Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM). Journal of Histochemistry and Cytochemistry. 
6. Yang X, et al. 2017. Optimizing Immunostaining of Enamel Matrix: Application of Sudan Black B and Minimization of False Positives from Normal Sera and IgGs. Frontiers of Physiology.  
7. Hoffman GE, et al. 2016. The Importance of Titrating Antibodies for Immunocytochemical Methods. Neuroscience. 
8. Buchwalow, I, et al. 2011. Non-specific binding of antibodies in immunohistochemistry: fallacies and facts. Nature. 
9. Mao S, et al. 2021. Blocking Cross-Species Secondary Binding When Performing Double Immunostaining with Mouse and Rat Primary Antibodies. Frontiers of Neuroscience. 
10. Wang H, et al. 1999. Detection of endogenous biotin in various tissues: novel functions in the hippocampus and implications for its use in avidin-biotin technology. Cell and Tissue Research. 
11. Grahek, M, et al. 2017. High-Sensitivity IHC Detection of Phosphorylated p27/Kip1 in Human Tissues Using Secondary Antibody Conjugated to Polymer-HRP. Methods in Molecular Biology.