TNFα Induced Cell Death in Skin Cells: possible role in Diabetic Wound Healing

Cell death was evaluated with DAPI staining. For DAPI staining cells were fixed to preserve normal cell structure and permeabilized to permit the antibody entry into the cells.
Cells were incubated with antibodies that target specific peptides or protein antigens in the cell via specific epitopes. Usually, after incubation with the primary antibody, cells are incubated with a secondary antibody, conjugated with a fluorofore, that binds it to the primary antibody.

Moreover, cells were stained with 4',6-diamidino-2-phenylindole (DAPI), which is a blue fluorescent probe that fluoresces brightly upon selectively binding to the minor groove of double stranded DNA (Kapuscinski et al., 1995).

After incubation, cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde in PBS for 10min, and then permeabilized with 0.1% Triton X-100 in PBS containing 200 mM glycine, during 5min. After a blocking step with 1% BSA, for 30min, cells were incubated with Alexa Fluor 555 phalloidin antibody (1:500) for 30min at room temperature and washed three times with PBS.
Then, cells were incubated for 1min with DAPI (0.1 μg/ml in PBS).
For image acquisition, fluorescence labelling was visualized using a fluorescence microscope.