DAPI staining in fixed Drosophila Hemocytes - (Aug/05/2013 )

Hi everybody,

I work with hemocytes extracted from drosophila adult flies and I have a problem with my DAPI staining. When I look at the cells under the microscope most of them are shallow bluish and do not show a clear signal in the nucleus. some of them however do. My protocol is as follows:

1. Extraction of the hemocytes into Schneider + Fetal Bovine Serum (10%) media

2. Fixation with cooled Formaldehyde for 15 min (3.7%)

3. Permeabilization with Triton-x 100 for 10 min (0.1%)

4. Dapi staining for 10 min (1 µg/ml)

The obvious difference between my protocol and others I found on the web is that i do not wash in between the steps. This is because too many cells get lost during washing steps. The question is, if this is the reason fpr the poor dapi staining. Is it possible that the cells are "overfixed" due to the long exposure to Formaldehyde and that the nucleus is damaged? If thatw as the case would it be a possible solution to add the Formaldahyde, Triton-X and Dapi at the same time and incubate them together?

Many thank in advance!

Overexposure to FA usually increases the crosslinking between proteins. I don't know in your cells but in bacteria it can reduce the cell permeability. In this case you get over it with Triton.

Is it a DAPI commercial solution? All the solutions are buffered? DAPI is quite sensitive to pH, reducing its fluorescence when you get far from pH 7. At least I have the experience that this is true if the pH goes down.

Additionally, if the protocol says washing, do it, there is a good reason to remove the previous chemicals: avoid unwanted and unknown chemical reactions.

If you are concerned about losing so many cells, have you thought about doing it on poly-L-lysine coated coverslips?