I am wondering why my hepg2 cells nucleus looks fuzzy and unclear under the confocal?
Is this mycoplasma contamination?
I used Hoechst staining, 1:200 dilute with 3%BSA.
Dear Cy!
Please You look at the protocol:
Article Staining and High-Resolution Imaging of Three-Dimensional Or...
7. Add 250 µL of Hoechst 33342 (20 µM stock solution) diluted at 1:1000 in PB:1x PBS (1:10) solution, and incubate for another 2 h at 4 °C with gentle horizontal agitation (30-50 rpm)
8. Let the organoids sediment, and gently remove the solution containing secondary antibody + Hoechst 33342. Wash the organoids 5x in 1 mL of 1x PBS for 3 min per wash and then 2x in 1 mL of 1x PBS for 15 min per wash with gentle horizontal agitation (30-50 rpm). NOTE: It is crucial to extensively wash the samples to avoid background noise or loss of signal.
https://www.researchgate.net/topic/DAPI
Aparajita Lahree
“Bethany Unger Hoechst33342 is cell permeable and usually used in live cell imaging. However for long term imaging, the UV laser illumination creates a lot of photo-toxicity. Its recommended hence, to use far-red DNA dyes like To-pro3.
There may be contamination or the cells are losing their signal due to extensive light exposure. I would recommend troubleshooting by using new cell cultures and trying to find the contamination source. Also, do not expose the stained cells to light/the confocal for more than a few minutes. Hope this helps!
Dear Cy,
First, be sure that your cells are not contaminated by mycoplasma because the cytoplasm is not stained by HOECHST. Second, when you observe your stained cells by a conventional fluorescence microscopy, the light dispersion causes the nucleus seems as integrated round blue units. Whereas by using confocal fluorescence microscopy, the unfavorable light dispersion will be eliminated, hence you'll observe the stained nucleus with more details. Since the HOECHST is intercalated into the DNA, and regarding the fact that chromosomes don't occupy all the space of nucleoplasm, I think that your microscopic image is absolutely perfect.
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