I'm confused with my immunofluorescence result. I have mixed cells from mollusc mantle tissue. It was stained with protein pAb/FITC antibody. However, when I stained with Hoechst, those which gave a signal from FITC, had no signal against the Hoechst. Staining was done on live cells. Can anyone share advice/tips on why this is happening?
Hoechst does not penetrate well in the living cells, it stain mainly death cells.
We've always used DAPI to stain live cell nuclei... could try that as a control.
Ooo. I thought Hoechst is better than DAPI when staining live cells. The stain is new. Its in a droplet container, ready to use. never tried the stai on other sample though. I have another one, DAPI. Will try that one next. The thing is, most of the cell stained is similar. If the DNA is not dense enough, will these stainers still be able to stain the DNA?
I think you have to consider all the checks suggested above, ie (1) using a new aliquot Hoechst, (2) using a control tissue or cellular culture. Additionally, you should keep in mind the type of DNA dye (Hoechst 33258 or 33342, the last stains live cells). Membrane transporters of the ABC type located in the cell membrane (and widely distributed in eukaryotic cells) are able to export the dye once it enters the cell to protect it from damage in DNA.
Vega, Thanks for the suggestion. The cells that I'm working with is primary cells, and there are so little information about mollusc cell in the references. I will try to use DAPI with fix and permeabilize cells and see what we have. Thanks again.
I agree, fixing cells and DAPI is the best decision. DAPI do not require permeabilization, for many cells type only fixation is enough. For the Hoechst dye, you should used only Hoechst 33342 and consider the fact, that not all buffer-medium will allow cells to uptake this dye in the living cells. This may be one of the main reason why you do not see staining in you system. .
Hi guys, I repeat my experiment and this time, I fix and permeabilize my cells. Similar problem occurred. Some cells which is stain by my antibody, had no signal from DAPI. Some cells show DAPI signal all over the cells, but very weakly. But still, the cells stained have no DAPI signal. I repeated this 2-3 times already with other cell, no problem. So...What kind of cells/stage in cell life that Its nucleus cannot be stained? I will try find any reference, if you have any, can you guys give the link. Thank you very much.
DAPI needs citric acid pre-treatment for binding DNA and is pH dependent http://botanika.biologija.org/exp/protocols/DAPI-staining-protocol.pdf
Hoeschst-33358 is non permeable in cells. Use Hoechst-33342 (this doesnt need citric acid treatment or strict pH and can be used in live cells too).
Hi, Fareed, you can find protocol for DAPI staining here: http://host.cc.ntu.edu.tw/biotech/Confocal%20Homepage/Nucleus.html. I am not sure that DAPI require citrate, even in the protocol mention by Goodwin, there is an option for DAPI staining only in PBS.
I would suggest you as the first control just fix, permeabilze and stain cells with 0.2 mg/l DAPI. Please, remember, that DAPI is very pH sensitive: only pH 7 is suitable. If pH of your buffer is not 7, (6.5-7.5 is OK as well) DAPI will stain all over the cells! Presence of some iones may also disturb DAPI staining. Good luck!
DAPI staining requires fixing and doubt if it could be used to stains live cells. However, I have used Hoescht 33342. It needs no fixing and it worked well for live cells.
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