Micronucleus assay to be carried out on fish.
I am attaching several images, Please tell us your opinion.
Hello Milad, it is possible to use Acridine orange staining and fluorescence microscopy. You should detect the frequency of micronucleated cells in the binucleated cell population.
Try these references:
Fenech, M. (2000). "The In Vitro Micronucleus Technique." Mutat Res 455(1-2):81-95.
OECD (2007). OECD Guideline for the Testing of Chemicals; Draft Proposal for a New Guideline 487: In Vitro Mammalian Cell Micronucleus Test (MNvit).
thanks marketa
but i carrying out on fish blood cells analyze and no mamalian cells.
Hello Milad,
My Observations based on the images you shared here....
1. It seems that your slides are not completely dried. Look at the cells, there is lack of complete absorption of the dye you used. It is due to the incomplete evaporationn of water content. After the smear preparation, keep the slides in absolute methanol for 10 min.
2. Left it for drying (Advisable to keep drying for 24 hours), preferably at 37 degrees.
3. Then follow a dual staining rather direct staining with Geimsa.
4. Stain the slides with May-Grunnwald stain (stain cytoplasm) for 10 mins and wash with distilled water and left air dry. Then stain with 5% Geimsa stain (stain nucleus) (Use your own lab prepared stain, If you use conventionally available stain, they wont retain the staining pattern for long run. this is from my own experience) for 10-15 mins.
5. Follow the above steps. You will get 100% success. Some of my own images for your reference
Dear Milad,
My experience is Giemsa staining and visualization with common microscope
Very good advice for me as well. Giemsa is very good, Acridine orange fades out after some time.
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