I'm planning to administer an immunogen labeled with AlexaFluor-488 into macaques with the goal to image the immunogen in the draining lymph node after it is biopsied. I'm having trouble finding papers where fluorescence-labeled proteins are imaged in fixed, paraffin-embedded tissues, and I am starting to wonder if there are issues related to the stability of this fluorophore that would prevent such an approach. Please let me know if you are familiar with any protocols where Alexa-Fluor (or similar probe) labeled proteins are imaged in formalin-fixed tissue, or what you think the best alternative for tissue preparation may be.
The paraffin embedding process will destroy your fluorescence: (from the Thermo Fisher website, “ Are Alexa Fluor® dyes compatible with common fixatives, buffers, permeabilizing agents like formaldehyde, formalin, paraffin, xylene, Triton® X-100, etc.?
Alexa Fluor® dyes are compatible with Triton®, formaldehyde, and formalin. Paraffin processing of tissues likely will extract dyes of almost any type, including Alexa Fluor® dyes, though they are fine to use after the processing, such as for immunohistochemistry (IHC) imaging.“ https://www.thermofisher.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/alexa-fluor/alexa-fluor-frequently-asked-questions.html
Have you considered frozen sections?
I would suggest you use fresh frozen or fixed frozen tissue for this purpose. Paraffin embedding is known to quench fluorescence present during the imbedding process.
Renee R Donahue Thank you Renee. I've decided to embed the tissues in OCT and have them cryosectioned rather than using paraffin.
- Badges
- Science topic
- Similar topics
- Computer Science
- Images