Immunofluorescence for detecting proteins in cytoplasm - (Apr/01/2014 )

Hi all,

I need some advice for a problem in Immunofluorescence.

 

I am interesting in a protein that is expressed mainly in cytoplasm. (as described in many references)

 

when I carried out Immunofluorescence to stain this protein, I found it was very bright in nucleus in most cells

 

I have searched this protein in google, and found most of the picture also showed a cytoplasm-located.

 

 

my protocols was as follows:

1 Grow  cells on  coverslips
2 Wash with PBS and fix with 4% PFA 1h at 4 degrees centigrade, followed by wash with PBS for three times

3 block and permeabilize with 0.1% Triton and 3% BSA in PBS for 1h RT
4 Incubate overnight with primary antibody, primary antibody was diluted in 3% BSA-PBS
5 Wash 3 times with PBS; incubate 3 hr RT with secondary; secondary was diluted in PBS
6 Wash 3 times and visualize

 

Did any procedures in the immunofluorescence affect the location of the protein?

I  have seen several posts on here, and I suspect the problem was in the fixation or the permeabilization. was the time too long?

 

could anybody shed light on this?

 

Any advice would be greatly appreciated!

There are any number of things that could be wrong here, part of the problem may be how you are looking at the cells - if you are not using confocal microscopy, then you are also looking at the cytoplasm that is above the nucleus.

 

1 hour fix is a very long time - try 10-20 min on ice. 

permeabilization for 10 min should be enough.

Try washing more - up to 6 x 5 min

3 hours is too long for secondaries, you have a very high chance of non-specific binding, try 1 hour at room temp.

 

Is the antibody you are using suitable for IF (is it the same one as in the literature)?  What do your antibody controls look like?  Do you have a negative and a positive sample you can test?

Hi Bob, Thanks very much for the advice!

I still have some questions about your response.

1. I used normal Fluorescence microscope not confocal microscopy to look at the cells, and I found the red fluorescence (I used Alexa Fluor 488 secondary antibody) was as the same position as the nucleus (I used Hoechst for nuclear staining). Does you mean that I can not  recognize the cytoplasm and the nucleus by normal Fluorescence microscope? The nucleus staining seemed very bright.

2. The instruction of the antibody said it was suitable for IF,and is used in a few literatures.

The protein is widely expressed in different kinds of cells. Does negative control mean that the sample not expressed the protein? That is a little difficult to obtain. 

 

Also I will try to reduce the fix, permeabilization and secondaries incubation time and do more washes. 

When you look at a cell down a microscope you are still looking at a 3D structure - kind of like if you crack an egg and spill out the contents onto a plate - the nucleus would be equivalent to the yolk.  So if you look down at the cell, the nucleus is below a small part of the cytoplasm that could easily be giving you some apparent nuclear signal, even if it isn't actually in the nucleus.  This is especially the case for peri-nuclear proteins (e.g. proteins bound to the nuclear membrane), as these will surround the nucleus quite tightly, and so look like nuclear staining.

 

If the antibody is used in the literature, it should be fine.  By negative control, I mean a cell type that doesn't express the protein, so that you can test the specificity of the antibody, it isn't absolutely necessary.

Thanks a lot. I understand what you mean. 

It seems looking at the cells by confocal microscopy is necessary.

I will do it again tomorrow.

 

I respectfully disagree. Regular Epi-fluorescent scope should be perfectly adequate for distinguishing between nuclear and cytoplasmic staining. I do agree that you should shorten fixation/staining times.  It would be helpful if you could post some of your photos as well as the name of the protein.

CellApplicationsInc on Wed Apr 2 18:48:41 2014 said:

I respectfully disagree. Regular Epi-fluorescent scope should be perfectly adequate for distinguishing between nuclear and cytoplasmic staining. I do agree that you should shorten fixation/staining times.  It would be helpful if you could post some of your photos as well as the name of the protein.

You are welcome to disagree - in most situations I agree that you can distinguish nuclear and cytoplasmic staining with an epifluorescent microscope, but I was talking about a certain subset of proteins that are strongly perinuclear (e.g. YB1), where you can't distinguish between nuclear staining and staining that closely surrounds the nucleus.