We are having problems staining nuclei of human osteoclasts. Dapi and Hoechst staining are too faint to discern clearly, and we are using the TRAP leukocyte stain (Sigma). Even after using tween 20 (0.2% 30min) still no DAPI/Hoechst. Any alternatives? I tried using H&E but the nuclei would not turn blue, suggestions?
Pictures are from 28 day culture, those look like nuclei and stain dark blue with Giemsa stain. When I combine the Giemsa with TRAP stain the Giemsa fails to label the nuclei clearly.
Hi Maria,
I am fixing cells with 5% gluteraldehyde for 10 minutes at room temperature. This is similar to what is recommended by the TRAP stain kit from Sigma.
Are you trying to stain nuclei with dapi and trap stain at same time? TRAP stain gives dark background under microscope when you visualize DAPI. Try Triton X-100 to permeabilize cells for DAPI. Perform hematoxylin based nuclear staining after you complete TRAP staining(30 sec-1 min). rinse the cells in tap water. Tap water is more alkaline than ph of hematoxylin and will give you blue nuclear staining after wash. Let the cells dry overnight and photograph next day.
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