I am currently working on an immunofluorescence experiment in spleen of mice in which I use an antibody conjugated to FITC. However, I am experiencing autofluorescence from red blood cells, which is interfering with the specific signal and reducing the clarity of my results.
I would greatly appreciate it if you could advise whether there are established methods to significantly reduce or eliminate RBC autofluorescence in this context. Have you had experience with this issue?
In addition, the use of sodium borohydride did not cause any significant change in autofluorescence.
Dear Dr. Fatemeh Alipour,
If it is possible for you, you may perfuse the spleen with PBS before fixation as it can help remove most of the RBCs and reduce their contribution to autofluorescence.
If you are using sodium borohydride, consider using increased concentration or extending the incubation time (up to 30 minutes) to see if that improves the results. The standard concentration of sodium borohydride for quenching is typically 0.1% or 0.3%. Also, the type of fixative used can impact the effectiveness of sodium borohydride. If you're using a strong aldehyde fixative, consider optimizing the fixation protocol.
If sodium borohydride isn't sufficient, consider using other quenching methods, either alone or in combination with sodium borohydride. Sudan Black B is a lipophilic dye that can effectively quench lipofuscin autofluorescence. I came across this study (article attached below) that used a combined application of sodium-borohydride followed by crystal violet before antigen retrieval and a Sudan black B treatment after secondary antibody application which proved to be most efficacious for masking autofluorescence /non-specific background in vascular tissues. You may try this trio method.
https://turkjpath.org/text.php?id=1843
Regards,
Malcolm Nobre
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides