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I currently work with fluorescence microscope to observe generative and vegetative cells within hibiscus rosa sinensis pollen. I used DAPI to stain pollens but there is no light that indicate the existence of both generative and vegetative cells. I simply stained the pollens with 50 ppb DAPI without other procedure. I have referred this steps from Sexual Plant Reproduction textbook from Cresti and Tiezzi, here is the link : le.ac.uk/bl/phh4/openpubs/SPR_HH_pollen_stigma_crestiLR.pdf. From the book, it simply states "For DAPI staining, fresh or fixed pollen is stained on a slide in a drop of 0.005% DAPI (diamidinophenylindole), and the nuclei observed under a fluorescent microscope after 5-30 min (UV excitation, blue DAPI fluorescence).

The result is make me confused because there was no fluorescent light but only reflected light by pollen itself.

I will be very glad if anyone can suggests me what should i do or highlights an error in my steps.

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  • $\begingroup$ What do you see after the staining? No light at all? How DAPI staining should allow you to distinguish between generative and vegetative cells? $\endgroup$ – LinuxBlanket Feb 7 '18 at 8:34
  • $\begingroup$ I saw light, certainly. But the light is light that was reflected by an object, not from the fluorescence phenomenon. I am not sure that DAPI can distinguish or make a different look for generative and vegetative cells because DAPI is only a compound that bind the A-T rich region and make the objects that were bounded by it able to emit a light due to fluorescence, not react specifically with generative or vegetative cells and make some different features (or another) between them. cmiiw. $\endgroup$ – Dziban N Feb 7 '18 at 10:19
  • $\begingroup$ So apparently the problem is that the staining itself didn't work at all. Probably the DAPI didn't penetrate in the cells. Did you solubilized the cell wall somehow? Can you detail all the procedure you followed prior to DAPI staining? $\endgroup$ – LinuxBlanket Feb 7 '18 at 10:39
  • $\begingroup$ Yes sure, I did it very simple (according to my reference (the textbook that i mentioned before) and suggestion from my lecturer). I just poured the pollens from the Hibiscus r. anther above the aquabidest at specimen glass, then dropped 50 ppb of DAPI above it and cover it with coverglass. And i think i miss the step of solubilizing the cell wall. Would you mind telling me how the protocol that should I do to solubilizing the cell wall of Hibiscus pollen? Or there is any reference that i can refer to do that? $\endgroup$ – Dziban N Feb 7 '18 at 10:57
  • $\begingroup$ Actually, this is only a step that my lecturer suggests to check whether or not my DAPI still viable to emit a fluorescence light. The core of my research is to staining a chromosomes with DAPI and observe its image under the microscope using a photonics jet phenomenon. $\endgroup$ – Dziban N Feb 7 '18 at 11:00

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