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Immunofluorescence (Immunocytochemistry)

Immunofluorescent analysis of HeLa cells using COX IV (3E11) Rabbit mAb #4850 (green), β-Catenin (L54E2) Mouse mAb (IF Preferred) #2677 (red) and DAPI (blue).

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Immunofluorescence (Immunocytochemistry)

Immunofluorescent analysis of HUVE cells using VE-Cadherin (D87F2) XP® Rabbit mAb #2500 (green) and DAPI (blue). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red).

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Product Description

DAPI is supplied as a lyophilized powder in 1 mg units. It can be used to examine cellular DNA in fluorescent microscopy and cytometry applications.


Molecular Formula:

C16H15N5 2(HCl)


Fluorescent Properties

  • Free dye excitation maximum = 340 nm
  • Free dye emission maximum = 488 nm
  • DNA complex excitation maximum = 364 nm
  • DNA complex emission maximum = 454 nm

Product Usage Information

Directions for Use: DAPI is supplied as a lyophilized powder. For a 20 mg/ml stock, reconstitute the 1 mg in 50 µl deionized water or dimethylformimide. Please follow CST’s recommended IF and Flow protocols. For both applications, following secondary detection:

Immunofluorescence: Counterstain with DAPI as the final step in your staining procedure. Rinse samples twice in PBS for five min each. Dilute DAPI stock solution to a concentration between 1 - 0.1 µg/ml in PBS and incubate for 5 min at room temperature in the dark. Rinse samples once in PBS and then prepare for imaging. Examine immediately using appropriate excitation wavelength.

Alternatively, dilute DAPI stock solution to a concentration between 1 - 0.1 µg/ml in mounting media, apply to cells, and prepare for imaging. Examine immediately using appropriate excitation wavelength.

Flow Cytometry: Rinse samples once in Incubation Buffer. Dilute DAPI stock solution to a concentration between 1.60-0.0125 µg/ml in PBS and incubate for 15 min at room temperature in the dark before analyzing cells on flow cytometer.


Storage: Storage lyophilized or in solution at 4°C, desiccated. Protect from light. In lyophilized form, the chemical is stable for 24 months. Once in solution, use within 2-3 weeks to prevent loss of potency. Aliquot to avoid multiple freeze/thaw cycles.

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).


1.  Portugal, J. and Waring, M.J. (1988) Biochim Biophys Acta 949, 158-68.

2.  Kapuscinski, J. (1995) Biotech Histochem 70, 220-33.

3.  Manzini, G. et al. (1983) Nucleic Acids Res 11, 8861-76.



For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
XP® is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor® is a registered trademark of Life Technologies Corporation.
DyLight™ is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.