Imaging fixed cells - Immunofluorescence (Jun/29/2006 )

Hello all!

I am trying to see were is my endogenous protein is located in the cell.
I'm doing a usual immunofluorescence: Fixation (PFA)--> permeabilization (TritonX)--> Blocking (FBS)--> 1st Ab--> washes (PBST)--> 2nd Ab--> washes....
My first Ab is Rabbit and my second is D-anti-Rabbit conjugated to RRX.
When I'm visualizing the samples using deltavision system (fluorescence microscopy) I can see the cells---- BUT!!!---- when I'm changing the wheel for filters that exites and emittes FITC I also can see the cells.... and this troubles me alot!
If I'm staining with RRX I shouldn't get any exitation using waveleangh suitted for FITC!
My lab friend told me that the cells themselves have an outofluorescence, but it doesn't make sense to me, otherwise all this method is inrelaible....! Now I'm wondering whether the images I get with the RRX filtered R true images of my endogenous protein or R they just an artifact?

I would apriciate any coment

Hi,

the cells are aotufluorescent but not so much to give you problems, unless you are trying to quantify the fluorescence with a program like ImageQuant (I had a lot of problems with that program). Can it be it's located all over the cell? Other question is you are washing with PBST: maybe you are permeabilizing too much your cells and what you see could be a too high background; I would use PBS or PBS++ for the washings

I'm washing with PBST (Tween 0.2%), which is the minimal perc. of tween. I have to have tween in the washing to get rid of unspecific binding of the antibodies....
But, my problem is that I'm using RRX, and when I'm visualizing the cells with waveleangh that are suitted for FITC (see, I want to see that I don't get faulse positives) I can see the cells almost the same as when I'm using wavelenght that are suitted for RRX exitaion....

Verify that the conjugate is RRX.

Try to look at the negative control, i.e. not using any antibody at all and only that the cells undergo the same treatment without any antibody (primary or secondary).

Also look at the cells treated with only secondary antibody.

This may help u identify the problem.

Dear Yaarit,

I think what are you seeing is a common problem with fluorescence. The EMMISSION spectra for both FITC and RRX overlap. Thus when you change the filter wheels you will see green fluorescence due to this overlap. I train people in my department in the art of Confocal Microscopy where we have the same problem. When exciting your sample with your light source or in our case a laser light, the overlapping emmission spectra's give rise to spurious fluorescence where you do not expect to see it.

I hope this is clear

Good luck

Rhombus

Thank u for your answers... Scolix, I will try fixing the cells without any antibodies, and see if the cells are truely so auto- fluerescence...
Rhombus, I'm glad you said what you said, because I suspected for along time that I am getting an overlap, but people told me that it is just can't be, otherwise all the assays that are trying to test for localization are miss leading. because if you have a red staining of you protein in the nucleus for example, and you also visualizaing another protein using FITC-conjugating antibodies, you might think that these 2 proteins are co-localized in the nucleus, where in fact you are getting a green staining due to the overlap from the red staining and not a true staining of your second protein......
What do you think of that?

Dear Yaarit,

When loooking for co-localisation using Confocal Microscopy, we have to eliminate the possibility of overlapping of the emmission spectra by doing SEQUENTIAL SCANNING. This involves exciting the sample with the green laser (FITC channel) and capturing the image, then switching the red (TRITC channel) on and capturing the image. The 2 images can then be merged by the software to achieve a TRUE image. If co-localisation exists, a yellow part of the image will be seen.
If you scan the image using BOTH lasers at the same time, then this will give rise to FALSE co-localised images because of the overlapping emmission spectra.
I have reviewed quite a few papers where it is obvious that false imaging has been done due to the ignorance of the researchers who use this powerful tool (confocal) in the wrong way. If you have a confocal locally, talk to the person who runs the machine, and he/she should be easily be able to demonstrate this effect.
NOTE : when choosing conjugated fluorophores for immuno's, check the emmission spectra's and try to eliminate the cross over by choosing 2 flourophores which have distinct spectra's.

Kindest reagrds and good luck

Rhombus