Immunofluorescence (IF) is an immunochemical staining technique that utilizes fluorochrome-labeled antibodies for the visualization and localization of targets, by labelling with a fluorescent dye such as fluorescein isothiocyanate (FITC). It can identify a wide range of components on tissue sections, cultured cells, or individual cells, and is thus highly valuable for their broad capability with the use of combinations of specific antibodies tagged with fluorophores.
There are several steps for IF, with the first being fixation to immobilize target antigens while maintaining cellular architecture. Next, sample preparation and blocking needs to be carried out to prevent non-specific binding of antibodies. Then, incubation of primary and secondary antibodies take place before the final preservation and imaging.
There are two different IF methods available: Direct (Primary) and Indirect (Secondary). Direct detection requires using a single fluorochrome-conjugated primary antibody. Meanwhile, indirect detection uses two antibodies, with the first being an unlabeled primary antibody, followed by a fluorochrome-conjugated secondary antibody.
Although both monoclonal and polyclonal antibodies can be used for IF, they have their own pros and cons. Indirect IF more commonly used as the secondary antibodies are typically polyclonal, allowing for the recognition of multiple epitopes of the targets. However, monoclonal antibodies which are specifically designed for IF may be more preferable if your research involves recognizing very specific targets.1
At Biointron, we are dedicated to accelerating your antibody discovery, optimization, and production needs. Our team of experts can provide customized solutions that meet your specific research needs. Contact us to learn more about our services and how we can help accelerate your research and drug development projects.
References:
Im, K., Mareninov, S., Diaz, M. F. P., & Yong, W. H. (2019). An introduction to Performing Immunofluorescence Staining. Methods in Molecular Biology (Clifton, N.J.), 1897, 299. https://doi.org/10.1007/978-1-4939-8935-5_26
Antibodies are versatile molecules that perform a range of effector functions, many of which engage different arms of the immune system. Their modes of action extend beyond simple antigen binding, enabling the activation of various immune mechanisms that lead to pathogen neutralization and clearance. These functions include blocking molecular interactions, activating the complement system, and linking the humoral immune response to cellular immune responses via Fc receptor engagement.
In today’s competitive biotech landscape, intellectual property (IP) protection has become an essential pillar in fostering innovation and collaboration across drug discovery and development. By offering clear IP terms and no royalty fees,pharmaceutical companies and research institutes
In addition to isotypes and subtypes, antibodies exhibit genetic variation known as allotypes, which are polymorphic epitopes on immunoglobulins. These allotypic differences arise from allelic variations in immunoglobulin genes, causing certain antibody subtypes to differ between individuals or ethnic groups. The presence of these polymorphic forms can influence immune responses, particularly when an individual is exposed to a non-self allotype, potentially triggering an anti-allotype immune reaction.
In mammals, antibodies are classified into five major isotypes: IgA, IgD, IgE, IgG, and IgM. Each isotype is defined by the heavy chain it contains: alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM). These structural differences in the heavy chains determine the antibody's function, tissue localization, and role in the immune response. Furthermore, antibody light chains fall into two classes—kappa and lambda—with kappa being more common, though both exhibit similar functions despite differences in sequence.
