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Antibody Selection for ICC/IF

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Fluorochromes Selection for Multicolor ICC/IF

Sample Preparation for ICC/IF Experiments

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Blocking for ICC-IF Assay

Antibody Selection in ICC/IF

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Secondary Antibodies

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ICC Handbook

Antibody Selection for ICC/IF

It is critical to select antibodies that are specific to the antigen of interest. Selecting the optimal antibody should include a review of available validation data, peer reviewed product citations, and end user feedback/ reviews. A low-affinity antibody may compromise staining, while a cross-reactive antibody may result in non-specific background or a false positive result. The figure on the right shows a side-by-side comparison of Novus’ beta-actin antibody (NBP1-47423) with a leading competitor. The diffuse signal shown by the competitor’s antibody is non-specific background.

ICC/IF of Histone H3 antibody

ICC/IF analysis of beta-actin in NIH-3T3 mouse embryonic cells using Novus Biologicals’ NBP1-47423 and a leading competitor’s antibody. The competitor’s antibody shows a high level of diffuse background/nonspecific staining. See more comparative testing examples at: Side-by-side comparisons of Bio-Techne and Santa Cruz antibodies

Critical Factors for Antibody Selection


Reactivity refers to the species of the antigen detected by a given antibody. When working in a human cell line, it is important to determine if the antibody has been tested against the human antigen. In some cases, a commercially available antibody that is reactive against the desired species may not be available. In this case, comparing the epitope sequence of the verified species to the sequence of the unverified species can predict antibody reactivity. An antibody may work in an unverified species if the epitope recognized by the antibody shares a high level of sequence similarity with the non-validated species. Novus guarantees that purchased antibodies will work in validated species and offers risk-free testing in unvalidated species.


Specificity refers to an antibody’s ability to recognize and bind a unique epitope on the target antigen. Antibody specificity is critical when detecting various post-translational modifications, sub-cellular domains or specific isoforms of a given protein. For example, detection of phospho-Ser10 Histone H3, a mitosis marker, requires an antibody that specifically recognizes Histone H3 protein only when it is phosphorylated at Serine 10 (See figure below).

ICC/IF of Histone H3 antibody

ICC/IF analysis of HeLa cells using Histone H3 (p Ser10] antibody [NB21-1091] with Alexa Fluor® 488- goat anti-rabbit IgG secondary antibody (green). Actin filaments were labeled with Alexa Fluor® 568 labeled phalloidin (red) and DAPI was used to stain the cell nuclei (blue).

*Note: Specificity of an antibody is often better confirmed with immunoassays other than ICC, such as Western blot and ELISA.


Antibodies are categorized as monoclonal and polyclonal based on the method of production and the number of epitopes detected. Key differences between monoclonal and polyclonal antibodies are listed below:




Production Produced from a single B cell clone. Produced from mixed or multiple B cell clones.
Number of Epitopes Detected Always detects a single epitope. Multiple when immunogen sequence is long or the full-length protein. Single in the case of small peptides or modification related antigens.
Cross-reactivity Highly specific, less likely to be cross-reactive unless the epitope is conserved among various species. More likely to be cross-reactive, especially antibodies generated against full length protein immunogens.
Working concentration 1-15ug/ml 5-25ug/ml


The host of an antibody refers to the species of the animal used to raise that antibody. In indirect detection, the host species of the primary antibody should be carefully considered. This is because each secondary antibody is directed against the species of the corresponding primary antibody (see figure below). Since more than one primary antibody is used in multicolor ICC, selecting primary antibodies raised in different species will minimize secondary antibody cross-reactivity and improve specificity. If the primary antibodies were both raised in the same species (mouse), then the corresponding secondary antibodies may bind to the primary antibodies indiscriminately. When the options of primary antibody species are limited, multiplexed immunoassays may use subclass specific primary and secondary antibodies (such as IgG1, IgG2b, IgG3, etc.).

Single antigen detection example: The mouse primary antibody requires an anti-mouse secondary antibody.

Multiple antigen detection example: The anti-mouse secondary antibody specifically binds to the mouse primary antibody, whereas the anti-rabbit secondary antibody specifically recognizes and binds to the rabbit primary antibody.

Multiple staining with primary antibodies raised in different species

Multiple staining with primary antibodies raised in different species for ICC/IF

See our Secondary Antibody Handbook for more information on staining with primary antibodies raised in the same species.

Application Compatibility

When choosing an antibody, confirm that the antibody under consideration is recommended for use in an ICC/IF assay. If options are limited or if the intention is to optimize an antibody which has not been validated in ICC/IF yet, try a few different conditions/ variables to see what works the best for generating accurate expression and localization data. In our experience, antibodies that detect protein in its native conformation are more likely to work in ICC/IF. Check out Novus Risk-Free Testing Program when purchasing primary antibodies in an untested species or application.

Concentration and Incubation Time

Some factors such as the working concentration of the antibody, antibody diluent, and incubation conditions (time and temperature) can affect antigen-antibody binding and non-specific binding. A high antibody concentration and a longer incubation time can result in overstaining cells. To determine the ideal antibody concentration and incubation time, review the recommendations provided in the antibody datasheet, as well as methods provided in peer-reviewed publications. When testing new antigens or non-validated antibodies, test a range of antibody concentrations, while maintaining the same incubation conditions (one hour at room temperature). Also, be sure to include the appropriate controls to validate the results.

Effect of different dilutions of the primary antibody on staining outcome

Staining outcomes for ICC/IF experiments

ICC/IF staining of cytokeratin in MCF7 cells using biotin conjugated pan Cytokeratin antibody (clone AE1 + AE3) [NBP2-33200B] followed by streptavidlin_Alexa Fluor 488. The data shown here is for different dilutions of the primary antibody: no primary control (A), 1:200 dilution (B), 1:400 dilution (C), and 1:800 dilution of NBP2-33200B (D). This data suggests 1:400 is the optimal dilution for this primary antibody under the tested conditions.

Antibody Wash Steps

Washing samples after an incubation with the primary or secondary antibody is necessary to remove the unbound antibody and to reduce non-specific antibody interactions. Inadequate washing may lead to non-specific background and overstaining of the cells. It is important to minimize the time between wash steps to keep cells from drying out and prevent staining artifacts. Samples incubated in wash buffer for an extended time can undergo cell lysis, despite the low concentration of the detergent in most wash buffers. We recommend 5-minute wash steps in PBS with 0.1% Triton X-100 or 0.05% Tween 20.