After treatment of cells with inhibitor and doxorubicin and doing confocal, I realise the doxorubicin stains both nucleus and cytoplasm making it difficult to get a good picture.
Hello Angelina,
Bleeding in cytoplasm is very common problem which many people encounters. Some easy steps can shoot this trouble very well. First of all you need to standardize your concentration for the cell number you are using. Use a lower concentration if cytoplasm spillage is a problem because high signal from nucleus bleeds to cytoplasm. So the main problem is with concentration. Same can be true with incubation time. Second, please take care of the washing steps. After incubation is over take care to wash the cells properly with wash buffer 3-4 times so that any non specific staining can be washed off. By following these steps, any fluorescent signal arising due to non specific binding will be automatically taken care of.
Hope it helps !
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