Chicken Polyclonals in Multiplex Immunofluorescence

Egg-celerate your multiplexing game using Aves Labs' chicken polyclonal antibodies!

When it comes to multiplexing, the more diverse the species, the better!

Multiplex immunofluorescence staining of three or more proteins in fixed cells or tissues can be challenging due to the lack of diversity in species used to produce commercial antibodies. Most antibodies are raised in either mouse or rabbit, so researchers are limited to probing only two proteins or cellular targets simultaneously.

That's why you need chicken antibodies!

Avian-derived antibodies are an excellent choice for complex multiplex IHC and ICC experiments with several protein targets as they show exceptionally high sensitivity and specificity, with no cross-reactivity to mammalian-derived antibodies.

Multiplex staining can be approached several different ways.

Continual advances in immunofluorescence techniques, fluorophore-conjugated antibody design, and complex microscopes now give researchers more tools than ever to pinpoint how the location of one protein in a cell or tissue relates to multiple other proteins simultaneously.

Direct immunofluorescence simplifies the staining process by directly conjugating primary antibodies to fluorophores with non-overlapping emission wavelengths and then imaged as different colors. However there is no guarantee that the protein target you are studying has a commercially available directly conjugated primary antibody.

Indirect immunofluorescence uses unconjugated primary antibodies raised in different species along with fluorophore-coupled secondary antibodies recognizing that particular species. In this technique however, researchers are limited by the species used to produce the primary antibody. Since primary antibodies are most often raised in mouse or rabbit, experiments can be limited to two proteins.

Unique species such as chicken allow researchers to perform three-color multiplex immunofluorescence experiments to get a clearer picture of multi-protein localization, potential interactions, and novel protein functions.

Chicken polyclonals are superior tools due to their unique immunoglobulins and evolutionary separation from mammals.

Chicken egg yolk contains only one class of immunoglobulin called IgY. Both IgY and its mammalian counterpart IgG (which has several isotypes) mount rapid responses to infectious agents but have subtle structural differences, meaning there is no cross-reactivity between antibodies from chickens and mammals. This makes them an excellent addition to multiplex immunofluorescence experiments.

Advantages of Chicken Polyclonal Antibodies

  1. Higher Avidity: Birds and mammals diverged around 300 million years ago, so they are quite evolutionarily distinct. This means that the chicken proteome is significantly different from the proteome of the commonly used mammalian antibody hosts rabbit and mouse. When developing antibodies to mammalian proteins, the immune system of rabbits and mice may have a less robust response because the antigen is being similar to their own proteins. A chicken's immune system reacts more strongly to these mammalian proteins because they are more foreign to them.
  2. Abundant and Convenient: One chicken can produce large quantities of IgY in egg yolk against low amounts of antigen. Immunized hens can lay on average 20 eggs a month with each yolk containing as much as 100 ug of IgY, a monthly yield that is equivalent to antibody derived from 300 mL of serum or 600 mL whole blood. Storage is also simple: chicken IgY is a thermally stable antibody and can be stored at 4°C for decades (or months at room temperature) without loss of activity. Storage is also simple as chicken antibodies are stable for months at room temperature, which will avoid any denaturing freeze-thaw cycles when storing in freezers.
  3. Humane and Non-Invasive: IgY antibodies are harvested only from eggs, so laying hens don’t undergo invasive blood harvesting procedures, reducing stress and increasing animal welfare, all with no compromise on the final antibody product. It is a win-win for both animals and researchers.
  4. No Cross-Reactivity: Avian IgY is distinct from mammalian IgG, so there is no cross-reactivity with mammalian-derived antibodies.
  5. Less Background: Chicken IgY antibodies lack an Fc domain, so they don’t recognize other aspects of the mammalian immune system, such as rheumatoid factors, complement, Fc-receptors, or proteins A or G. This means less background signal and fewer false positives in mammalian in vivo studies than with IgG-based antibodies. Using our highly cited BlokHen® blocking reagent can reduce background staining even further in applications such as immunocytochemistry, immunohistochemistry, ELISA, and Western blot.
  6. Compatible with IgG-based assays: Both IgG and IgY are functionally equivalent in most biochemical or cellular applications, so IgY can be used as a direct alternative to IgG in immunofluorescence, immunohistochemistry, immunocytochemistry, ELISA, Western blot, and more.

Try our chicken antibodies today to discover how chickens make better antibodies.