Hi,
I am using Acridine Orange staining to visualize autophagic cells. The staining should remain green in the nucleus (sometimes also in the cytoplasm) and turn orange within autophagic lysosomes, showing only orange dots in case of autophagy.
However, it seems that the orange coloring is the same and overlapping on the green one. Had this happened to anyone? How can I fixed the problem?
Also, I'm wondering if it is possible to fix with PFA 4% after staining.
Thank you.
Dear Gulia,
prior to help you I need following infos :
- Are you using A.O base or hydrochloride or hydrochloride with zinc chloride. Suppliers samples have a dye content between 80 % and 60% (C17H20ClN3 or C17H20ClN3 .ZnCl2).
-The concentration of your A.O dye is ...? for the staining.
-Buffer used e.g. pH of the buffer solution.
-Protocol used for the vital staining with A.O.
- If you use the A.O. ZnCl2 salt, ZnCl2 will be toxic for your cells according to the concentration.
Thank you in advance for your answer.
Good luck
Jean-Rene Grezes alias JRG
Good morning,
first of all, thank you for your interest in my question.
The acridine orange dye I am using is the product of Sigma A8097, i.e. A.O hydrochloride solution 10 mg/mL in H20 (without zinc).
For staining, I dilute the Sigma product 1:10.000 in PBS pH 7.4. Incubation for 15 min in the dark at 37°C with 5% CO2. Subsequently, 3 washes in PBS. Then, I tried to look under the microscope immediately without fixation, keeping the cells in culture media or in PBS pH 7.4. Or I tried to fix with 4% PFA for 5 min. In all these cases, my problem remains the same.
Thank you further.
Kind regards,
Giulia
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