Hi Mahdis Taghavibayat

Performing immunofluorescence (IF) on paraffin-embedded tissues does indeed increase the chances of encountering autofluorescence compared to frozen tissues. Here's why:

* Formalin fixation: Formalin, a common fixative used for paraffin embedding, can induce autofluorescence in tissues. It cross-links proteins, leading to the formation of fluorescent adducts that emit light when excited with the laser used in IF microscopy.

* Autofluorescent biomolecules: Paraffin itself can exhibit autofluorescence. Additionally, certain biomolecules naturally present in tissues, such as lipofuscin (wear-and-tear pigment), collagen, elastin, and red blood cells, can also increase autofluoresce.

The age of paraffin-embedded tissues can indeed affect the results of immunofluorescence (IF) and immunohistochemistry (IHC) in several ways.

* Antigen degradation: Over time, proteins and other target molecules (antigens) can degrade in paraffin-embedded tissues. This degradation reduces the number of available targets for antibodies to bind to, leading to weaker staining intensity and potentially reduced sensitivity of the technique.

* Reduced antigenicity: Formalin fixation, a key step in paraffin embedding, can mask some antigenic epitopes (binding sites) on proteins. Over time, these masked epitopes might become permanently unavailable for antibody binding, further compromising the staining results.

* Increased autofluorescence: Older tissues might exhibit higher levels of autofluorescence due to factors like further cross-linking of proteins by formalin and accumulation of autofluorescent biomolecules. This autofluorescence can mask the true signal from your target protein.

Please find the references attached.

* https://wellcomeopenresearch.org/articles/2-79

* Article Immunohistochemistry for Pathologists: Protocols, Pitfalls, and Tips

Thanks,

I agree with Samir. I just give you some pracrical recommandations, just in case you need to use old paraffin material.

1. use fresh solutions (alcohols and xyleen) for dewaxing and rehy3dration of the paraffin sections

2. depending of the antibodies you like to use most the time it is recommanded to do an antigene retrievel in mikrowave or steam cooker either in citrate buffer (pH 6) or EDTA buffer (pH9). In teh data sheet of your antibody there is mentioned which kind of buffer you should use

3. use positive controls of freshly embedded tissue (quality of the specific binding of the antibody) and negative controls of new and old paraffin sections to find out the grade of autofluorescence depending background staining

4. the autofluorescence is very strong in the 488nm channel. If it is possible use other channels like 647nm (Cy5) or 750nm (Cy7)

Ute Neubacher Samir Ranjan Panda Thank you for your responses. Do you think immunohistochemistry is more advantageous than immunofluorescence in paraffin-fixed tissue, even after antigen retrieval?

Well, I think any kind of immunhistochmical reactions, and immunfluorescence is a part of it, is a challenge especially when you have less expirience. Positive and negative controls could help to validate and verify your results. If you think twice you can also play with bright field techniques and immunfluorescence. That means if you have daubts about the specific binding of your Antibodies in immunfluorescence I recommend to do the the same reaction with a peroxidase labeled secondary antibody or streptavidin and afterwards detect the Immuno reaction with DAB. Under this circumstances it is much easier to distinguish specific labeling from unspecific background staining.

Ute Neubacher I sincerely appreciate your guidance; it has been exceedingly helpful.