Dear Dziban,

first, I think there´s a typo in your description: 50ppb would be to high diluted, according to mentioned protocol you should use 50 parts per million (ppm)!

For animal tissue, I use DAPI at 1 ppm (~360nM) which perfectly stains nuclei, but involves additional rinsing, because else some background fluorescence may impair the signal.

Second, I´m not familiar with plant tissues, but wouldn´t it be necessary to somehow treat the pollen with detergent to make the DNA accessible for DAPI?

Third, I miss your negative controll for autofluorescence. You should provide these pictures to allow comparison with the DAPI staining, i.e. untreated pollen under UV excitation/ DAPI filter, without transmission light. Autofluorescence is a common phenomenon, so don´t forget the negative control. To me, the structures don´t look like nuclei.

Hi Dziban,

As Christoph-Rüdiger says, you need a negative control.

The bottom picture looks like reflection to me. Not specific fluorescence.

Dear Dziban,

DAPI is always described as being semi-permeant, but in plant cells, internalisation of this dye is highly problemmatic. It selectively stains dsDNA, preferentially binding to AT clusters in the minor groove of the DNA molecule. On binding, the dye undergoes a 20-fold increase in fluorescence. The dilactate form of the dye may be more permeant than the standard, dihydrochloride form. DAPI can be used to show chromosome banding. It is unstable in light and should be stored in dark bottles.

Prepare a 0.5 mgml-1 aqueous stock solution and dilute this in water or buffer to give a final concentration of 1 µgml-1. DAPI is photo-labile and should be stored in brown glass or foil- covered bottles at 4°C. Stain tissue for 1 - 10 minutes before mounting on a microscope slide. If staining is poor, the tissue can be lightly abraded or vacuum infiltrated with the dye. The tissue can be rinsed and mounted in water, or mounted in dye solution to increase the staining. DAPI is subject to quenching after a short excitation period, causing rapid reduction of fluorescence with time and necessitating relatively fast image acquisition to maximise the fluorescent signal. To reduce this, samples can be mounted in an antifade mountant such as Citifluor, Dabco or Fluoromount.

Thanks for the answers Mr.von Bredow, Mr. Vlierberghe, and Mr. Aydin..

Actually, my primary research is about chromosomes observation using photonics jet (physics phenomenon that concentrate an intensity of light at a certain point) and the chromosomes that used for my research is human chromosomes that prepared for karyotyping.

The pollen staining is a suggestion from my lecturer to check whether DAPI is still viable to be used or not

The DAPI that used for counterstaining the chromosomes has a concentration 125 ug/ml. I have bought it from Abbott Molecular in amount 2 x 500 ml. Is this DAPI (specifically, it labelled as DAPI II Counterstain), whose concentration 125 ug/ml, able to staining the human chromosomes? Or the concentration of DAPI is too low?