I am currently tryig to do double immunfluorescent staining in myotubes but I could not be able to see a clear negative control.
As you may see in the presentation, I used two different kinds of negative control. 1) no primary antibody 2) Another cell line that should not be expressing desmin (fibroblast). In both of my negative controls I still have strong staining, especially localized in nuclei that I could not get rid of.
%4 PFA is my fixative agent. I am using donkey anti-rabbit (AF488) and donkey anti-goat (AF568) secondary antibodies in 1/5000 dilution.There is not any negative staining in Texas red but in FITC. I do all the washes with %0,5 PBS(T) and 15 minutes. I used 1% FBS and 5% BSA blocking but there was no change. Moreover, I changed the cell type and do the experiment in C2C12 myoblasts but still the same result. Finally, I did desmin staining on its own but negative control was not clear again with donkey anti-rabbit (AF488) but when we do double staining in skeletal muscle tissue, the result was satisfying; no staining observed at negative control.
What could have gone wrong with the cells or antibodies? Any ideas?
Thank you for your kind answers.
Dear Seyda,
One problem may be autofluorescence from Paraformaldehyde, which is typically seen in the FITC (488) channel. If you take some cells run them through your protocol, but do not apply any antibodies at all (either primary or secondary), do you still see a light green signal?
If so, there are methods to quench autofluorescence. I usually use 0.1 M glycine in PBS, pH 7.4, as a 30 minute rinse, before adding my blocking agent, on 11um-20um tissue sections. If you don't have any glycine, lysine works too.
Otherwise, try using a different wavelength fluorochrome. Jackson ImmunoResearch has a large variety of Donkey anti-rabbit secondaries that are useful for double-labeling (note: no financial interest).
Good Luck!
Jill
Hi Seyda, I agree with Jill about autofluorescence and using longer wavelength fluorophores.
Quenching autofluorescence can be done by incubating samples with primary amine buffers (glycine, tris, etc.) but if you want to do it quickly, you could use sodium borohydride (NABH4).
https://www.nature.com/articles/srep33488
I washed %0.2 glycine in PBS 2X5 minutes and 1X5 minutes PBS after PFA fixation than I did permeabilization and blocking.
I can try not applying any antibodies, I have not done that.
Thank you for your answers.