Dear all,
I am doing immunocytochemistry with HL-60 cells. I tried to detect LC3 puncta for autophagy investigation.
I used mounting media containing DAPI from Vectashield (aqueous form). All the buffers I used were filter through 0.22um nylon filters. However, after mounting the slides and storing at 4oC overnight, I observed lots of tiny fluorescent particles moving around the cells inside the mounted slides. I guessed my slides got contamination with bacteria.
I tried several times and got the same problems.
However, that contamination did not occur when I did experiments with human blood neutrophils.
Could you please suggest me the reason that could cause the problem and what I should do to avoid it?
Thank you so much for your help!
Using the mounting media containing DAPI, I would suggest putting a thin layer of clear nailpolish on the sides of the coverslip as to prevent the coverslip from moving and fixing it in place. This should prevent the moving of fluorescent particles. I hope it helps.
Thank you Kruger,
I though the problem was that I used the mounting solution which remained in liquid phase after mounting (did not get hard). May I ask which mounting solution you are using now?
Thank you again!
Hi Le-Duy,
Sometimes the antibody is contaminaited with debries so you can clear this by spinning them down.
good luck!
Dear Le-Duy
I also use the mounting solution that remain in liquid phase. That is why I use clear nail polish to fix the coverslip in place. That will prevent the coverslip from moving and therefore cause bubbles. Spinning the antibody before use can also be of help as Johan suggested although it will not prevent the moving of the coverslip and bubbles.
Hi Ley-Dy,
this sounds like you have a mycoplasm contamination in your cell line.
You should run a mycoplasm test before you continue your work with these cells.
DHi Le-Duy,
using nail polish is very useful as suggested by Maritza.
and always check for mycoplasm once in 3 months contamination before experiments!
Mycoplasma do not move, albeight blue on DAPI. It is in cells rather than out of cells after a lot of washing.
Hello all,
I used nail polish always after mounting the slides.
I tried to used harden mounting solution and did not observed the moving flourescent particles anymore. I think the from now on I will use harden mounting solution instead of aqueous-remaining solution,
Thank you all for your help.
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