Epifluorescent analysis of HeLa cells using COX IV (3E11) Rabbit mAb (Alexa Fluor® 488 Conjugate) #4853 (green) and ProLong® Gold Antifade Reagent with DAPI (blue pseudocolor).
Confocal immunofluorescent analysis of COS-7 cells, mounted in either glycerol (upper panels) or ProLong® Gold Antifade Reagent #9071 (lower panels), using β-Actin (8H10D10) Mouse mAb #3700 and Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 555 Conjugate) #4409 (red) (0, 3.3, or 10 min of imaging).
Following immunostaining, cover samples with ProLong® Gold Antifade Reagent with DAPI #8961 and coverslip.
To prevent fading, allow slides to cure in the dark, at room temperature for 24 hours, then seal the coverslips with clear lacquer, such as nail polish.
Proceed with imaging.
Store at -20°C. ProLong® Gold Antifade Reagent is stable for 12 months when stored protected from light, at or below -20°C.
ProLong® Gold Antifade Reagent with DAPI offers enhanced resistance to photobleaching, is premixed and ready to use, and causes little or no quenching of the fluorescent signal. The reagent cures within 24 hours and samples can be saved for months after mounting. This reagent is compatible with most dyes and is appropriate for use in multi-color labeling assays.
The inclusion of DAPI within this mounting reagent eliminates the need for a separate nuclear counterstaining step. 4', 6-diamidino-2-phenylindole, dihydrochloride (DAPI) is a blue fluorescent DNA dye that targets double-stranded AT clusters in the DNA minor groove (1). One molecule of dye binds to each 3 base pairs of dsDNA and yields an approximate 20-fold fluorescent enhancement (2). The level of DAPI-DNA fluorescence is proportional to DNA content (3).
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