I am trying out immunofluorescence on rabbit spleen but the results are not fruitful due to auto-fluorescence. I tried blocking with 5%BSA for an hour still persists. I also tried sudan black but could not see any signal, even in the positive control. Maybe i am doing it wrong.
Help from the experts in the field is much appreciated.
Thanks in advance.
Dear Narendran,
Indeed autofluorescence can be a drag and some tissues are worse than other. I guess lots of autofluorescence in spleen comes from haemoglobin. If you have the chance to use confocal laser scanning microscope than the lambda scan over the whole wavelength range can help you determine which wavelengths emit the least autofluorescence and than just hope that your specific signal would be higher. This way you can define the signal treshold on the negative sample and without changing any acquisition settings collect the positive signal above the treshold from your samples.
Hope this helps. Good luck.
Sasa Stefanic thank you very much. Unfortunately, the problem here again is that, the autofluorescence is higher than the the specific signal we are looking for.
@kaushiki Chatterjee thank you very much. Is there any way to differentiate between autofluorescence and non-specific binding. Because a slide without primary antibody and only secondary antibody gave similar fluorescence.
@kaushiki Chatterjee, I will try doing as advised by you... Thank you very much
Hi Narendran
You can also try these two techniques to reduce auto fluorescence.
1. Use Tween 20 instead of Triton in blocking buffer
2. Use the serum of same species of your secondary antibody, like if you are using goat anti-rabbit secondary antibody, use goat serum for blocking.
Good luck
You can try using 10% normal serum in your blocking step instead of using BSA, it reduces auto fluorescence significantly.
Narendran Reguraman The type of serum depends on the animal where secondary antibody is raised. For example if i am using secondary antibody like Donkey anti rabbit then i will use donkey serum, if secondary is made from goat then i will use goat serum.
- Badges
- Science method