I had recently encountered a strange problem while imaging using the confocal. The cells labelled with GFP (lentiviral) were transplanted into the animal. After 2 months, the animal was sacrificed and the transplanted region was removed and fixed in formalin followed by decal. Sections were taken using the microtome, after which I added the coverslip and proceeded for routine imaging using confocal.
The problem is:
All the cells are showing positive in Alexa 488 range, EGFP laser and 594 laser along with which DAPI staining is also seen. But am very sure that I haven't added DAPI or rather any other staining in it. The nucleus looks quiet conspicuous and I am not all sure how the cells are positive for DAPI staining. Not only the sections which has GFP showing this kind of problem but also the cells which are acting as a control (no GFP tagging) is also showing similar expression pattern. I checked for background noise and it is not falling in that range too. How is it possible? I'm very confused with the results.
Hi Sowmya,
Check out this discussion:
https://www.researchgate.net/post/GFP_fluorescence_after_fixation10
You might find a good answer there.
Best,
Kai
Hi Sowmya,
1) Did you verify that your lentivirally generated cells DO express the GFP-construct before you transplanted the cells into mice?
2) How did you see the GFP-fluorescence in those cells? Live or fixed?
3) If you only checked the expression live (or on WB), this could point to a fixation problem and the signals that you see are only background fluorescence.
4) Try to stain with an antibody against GFP (labelled for ex. with Al488) to boost your GFP signal.
Good luck and keep us posted!
Eva
Thanks Eva.
1. Yes, I verified the expression of GFP (live cells) before the transplantation.
2. The tibia from the transplanted region (after 2 months) was removed and it was fixed in formalin. I checked for the GFP expression after taking the sections (without staining) on the slides (fixed tissue)
3. The images are so deceiving that it looks like a true stain.
4. I stained the sections with GFP (primary) and a 594 conjugated secondary. I think I did a mistake by not getting a 488 thinking that it might lead to false positive result.
I shall do that.
Sowmya
Dear Sowmya,
it seems that you have deal with autofluorescence.
What I would suggest would be to stain cells independently with each dye, 488, 390/405 (DAPI), 594 in order to analyze these spectra by confocal microscopy. Record these spectra by carrying out an ²lambda scan". Take the 488 laser and a resolution of 512x512 pixel, 594 and DAPI the same.
Then take your animal sample. Carry out the same lambda scans and compare the results.
If you obtain distinct peaks as you were expecting them, then there is a different problem. If you'll get a signal over throughout the whole spectra, then you you' ll experience autofluorescence. Very often liver section will give you autofluorescence.
I hope this will help somewhat.
Ingo
Hi Sowmya,
With question 2) I was referring to the stable cell line that you created and not to the mouse transplants. You wrote in 1) that you looked at the cells before transplantation LIVE. This indeed points to the fixation beeing the problem and what remains to be seen is autofluorescence. Besides the "boosting" with the Al488, you could also have another look at your stable cell lines and see whether you loose the fluorescence upon fixation and whether the anti-GFP (Al488) solves the problem. To check easily whether a signal is autofluorescence or real, you could mix your GFP-line with a non-fluorescent or preferentially a red-fluorescent line. Then you should be able to easily distinguish the GFP-positive cells to the non-GFP-fluorescent ones. This will also give you a feeling of how autofluorescence looks in your cells (but unfortunately not in tissue).
Good Luck!
Eva
HI was your problem solved ? I am having a similar issue with my GFP mice. The thing which is bugging me is the DAPI stained cells. I didn't stain the tissue with anything close to DAPi and i still see DAPI positive nucleus staining.
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