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Detecting Chicken IgY in Immunohistochemistry

If you're working on mammalian proteins essential for life, IgY might be the tool you're looking for. 

Detecting Chicken IgY in Immunohistochemistry and Immunofluorescence.

Chicken IgY is increasing in popularity, since chickens are far removed from mammalian species and mount a vigorous immune response to mammalian proteins, enabling antibody production to some essential proteins that cannot be raised in mammalian species.  IgY also has an advantage that it is  too dissimilar from IgG to cause cross-reactivity issues. IgY can be purified from eggs, as they do at Aves Labs, not requiring serum collection, a much easier process for the animals.

Chicken Primary Antibodies

Although there are many off the shelf IHC detection kits for detecting primary antibodies from a range of species, these kits are scarce for Chicken IgY. We’ve assembled a suggested protocol and products so you can use IgY antibodies with confidence.

Direct conjugates

Directly labelled anti-chicken can be the simplest way to detect the Chicken IgY, however, as this is not as sensitive as other methods, the concentration of the primary antibody may need to be relatively high. 

These can be applied to immunohistochemistry/ immunofluorescence, blotting and ELISA (direct) methods. On prepared and blocked tissues/ samples, incubate with the primary, wash, incubate with the secondary, develop as required and mount.

HRP conjugated   AP conjugated   Fluorescent conjugates

 

Immunohistochemistry – ABC method

                  

AVES CNPase on mouse brain Cat# CNP

Incubations at room temperature unless stated

  1. Deparaffinise sections and bring to water if paraffin section being used. Fix frozen sections if necessary
  2. Wash 5 minutes in tap water
  3. Antigen Unmasking/ epitope retrieval if required. Read More 
  4. Quench endogenous enzymes if needed - 10 minutes with a reagent such as BLOXALL®
  5. Wash in buffer for 5 minutes
  6. Serum block – 20 minutes, ideally in diluted serum from the species in which the secondary antibody was made, an Animal Free Blocking Solution , or BlokHen. If biotin blocking is required, add the avidin of blocking kit to this step
  7. Drain excess from slide
  8. Chicken IgY primary antibody incubation – for determined optimal time, temperature and dilution of incubation. If biotin blocking required, add biotin of blocking kit to this step. Dilution buffers may benefit from 2.5% blocking serum
  9. Wash 5 minutes in buffer
  10. Secondary antibody - Incubate for 30 minutes with diluted Biotinylated Anti-Chicken IgY
  11. Wash 5 minutes in buffer
  12. ABC detection – 30 minutes with prepared ABC kit. (These kits are all ABC reagent only )
  13. Wash 5 minutes in buffer
  14. Develop with an appropriate precipitating substrate to the reporting enzyme chosen. Follow kit instructions for desired intensity.
  15. Rinse in tap water
  16. Counterstain if desired. Read More
  17. Clear & mount or mount as appropriate to substrate requirements. Mounting Media  

 

 

Immunofluorescence

A secondary directly conjugated to a fluorophore can be used as the secondary antibody, then wash and mount. This is a shorter, less sensitive method.

Fluorescent conjugates

For an amplified method using biotin

  1. Deparaffinise sections* and bring to water if paraffin section being used. Fix frozen sections if necessary
  2. Wash 5 minutes in tap water
  3. Antigen Unmasking/ epitope retrieval if required. Read More 
  4. Serum block – 20 minutes, ideally in diluted serum from the species in which the secondary antibody was made, an Animal Free Blocking Solution , or BlokHen . If biotin blocking is required, add the avidin of blocking kit to this step
  5. Drain excess from slide
  6. Chicken IgY primary antibody incubation – for determined optimal time, temperature and dilution of incubation. If biotin blocking required, add biotin of blocking kit to this step. Dilution buffers may benefit from 2.5% blocking serum
  7. Wash 5 minutes in buffer
  8. Secondary antibody - Incubate for 30 minutes with diluted Biotinylated Anti-Chicken IgY
  9. Wash 5 minutes in buffer
  10. (Strept)avidin detection – 30 minutes with diluted Avidin or Streptavidin fluorophore conjugate
  11. Wash 5 minutes in buffer
  12. Counterstain if desired with fluorescence counterstain and mount, or mount in anti-fade mounting media with or without counterstain

 

*Formalin fixation can cause autofluorescence. Some tissue elements may also autofluoresce. If controls indicate this is an issue, consider using an autofluorescence quenching reagent such as TrueVIEW for non-lipofuscin sources. Quenching this autofluorescence may mean the stain needs optimisation in conjunction with the quenching protocol. Check an unstained section under the fluorescent microscope when you’re planning your method, so you only have to optimise your staining once! 

 

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