Immunolabeling – using Primary and Secondary Antibodies

Immunolabeling or immunostaining is a method that is extensively used in molecular biology and biochemistry. It uses binding of antibodies to detect antigens on specific cells/tissues/organs. I use this technique a lot in my lab when I want to do some neuron tracing in my virus-injected free-floating brain sections. Well, then what are antigens and antibodies?

Antigens are molecules (can be nucleic acids/polysaccharides/lipids/proteins) that initiate the production of antibodies. Foreign antigens originate from foreign bodies such as virus, bacteria, and protozoa. On the other hand – DNA, enzymes, proteins produced by your body are known as self-antigens.

Antibodies are what are required for you to recover from a fever or a cold, i.e. for creating an immune response. An epitope is a part of the antigen to which the antibody attaches itself. There are two groups of antibodies –

  1. Primary (or 1 degree) Antibodies – it directly binds to a protein/enzyme i.e. an antigen.
  2. Secondary (or 2 degree) Antibodies – this binds to the primary antibody (which binds to an antigen), hence it is said to have an indirect connection. Some conjugated secondary antibodies are known to have ‘fluorophores’ (for immunofluorescence) on them. Each of these conjugated antibodies have 2-7 fluorophores on them, these enable us to see parts of the tissue/cell that is expressing the antigen of interest. (Check out ‘Cool Pictures from the Lab’ section of my website to see some immunostained brain sections!)

Primary and Secondary antibodies used in a laboratory setting for immunolabeling are from hosts (i.e. animal species) different from the sample species. This is to avoid cross reactivity between the two samples. Let’s say you are working on a mouse specimen, then it is important to use primary antibodies from animals other than mice such as goat, rabbit, donkey, chicken, etc. Secondary antibody is picked based on the host of the primary antibody used. For example, if your primary antibody was a chicken monoclonal antibody, your secondary will be anti-chicken secondary.

Various immunolabeling experiments can be set up once you properly understand the mechanism of antigen-antibody binding. Double labeling means using two primary antibodies and two respective secondary antibodies to label two proteins or enzymes. Triple labeling means using three of both primary and respective secondary antibodies. Immunocytochemistry (ICC) is targeting and staining cells, and immunohistochemistry (IHC) is targeting and staining tissue antigens.

Scientifica. 2021. #LabHacks: What you need to know about immunolabeling procedures. [online] Available at: <;

Self antigens. 2004. Moreland L.W. (eds) Rheumatology and Immunology Therapy. Springer, Berlin, Heidelberg. [online] Available at: <; 2021. Choosing an antibody | Abcam. [online] Available at: <,endogenous%20immunoglobulins%20in%20the%20sample.&gt;