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!
Edited by TimP, 05 August 2013 - 04:56 AM.