DAPI staining of paraffin sections

DAPI (4′,6-diamidino-2-phenylindole) is a fluorochrome that strongly binds with the minor groove of DNA with a preference for the adenine – thymine base pairs. When bound to double – stranded DNA, DAPI has an absorption maximum at a wavelength of 358 nm (UV) and its emission maximum at 461 nm (blue). It can also bind to RNA probably at the level of adenine – uracil clusters. The DAPI/ RNA complex has an emission maximum at 500 nm a longer wavelength than for the complex DAPI/DNA and with a lower fluorescence.

Protocol for paraffin section (8 μm thick)

  • Deparaffinize and hydrate to water.
  • Use a pen to draw a hydrophobic circle around sections
  • Stain with DAPI at appropriate concentration varying from 0.1 mg/ml to 1 mg/ml.
  • Add a drop of DAPI on the sections.
  • Wash 3 x 5 min in distilled water
  • Mount in appropriating medium such as Vectashied
  • Observe with a fluorescent microscope using appropriate filters. 
    For Inverted Fluorescent Microscope Leica DM IRE 2 the cube filters used contains an excitation filter in UV (340 – 380 nm); a 400 nm dichroic mirror and an emission filter or barrier filter LP 425 nm.

Below : Parts of paraffin sections through a primary Zea mays root tip

Mitosis (white arrow) are clearly seen.

Below : Parts of paraffin sections through root nodules induced by the bacterium strain NGR234