Colocalization of alexa 647 - (Jun/24/2007 )
hello,
i did a immunohistochem on kidney section staining for GFP using rabbit IgG as primary Ab and alexa 647 as sec Ab. when i am viewing the slides, i realised that i only see random staining like red stains on the surface or in between cells. how do i see co localization of the stain?? if it is suppose to be within the cell, why is that when i perform Z sectioning, i am not able to see the stain that i saw withing the cell before. it just disappears.
i did a immunohistochem on kidney section staining for GFP using rabbit IgG as primary Ab and alexa 647 as sec Ab. when i am viewing the slides, i realised that i only see random staining like red stains on the surface or in between cells. how do i see co localization of the stain?? if it is suppose to be within the cell, why is that when i perform Z sectioning, i am not able to see the stain that i saw withing the cell before. it just disappears.
Could you please explain your question (and your process) a bit more? I am not very sure what you are talking about. GFP is green, alexafluor 647 belongs to the FarRed range (that is, can't see by your eyes under microscope thus only machine can capture it for you. The default setting colour for it is usually blue. But you are talking about red???? Normal rabbit IgG should not stain anything in this case, should it? Or are you talking about rabbit anti-GFP, for example?
My main objective here is to check for the presence of EGFP positivity in kidney cells of maternal organs deriving from mice. thus i am using rabbit anti -GFP as primary Ab and alexa 647 goat anti rabbit for secondary staining. yes, i am using a confocal microscope to view my slides. I have changed my viewing colour for alexa 647 to red as i have stained my sample with DAPI which is blue. the problem i am facing now is that i am unable to conclude if there is co localization of the alexa 647 on the kidney cells. it is because when i do see stains inside the cells, i perform a Z (depth) viewing of 0.5 step size and 20 slices of the images. however after that, i see that the staining disappears as the images proceed towards the end. like i see the stain from 6-12 of the images but after that i only see the dapi stain of the cell. or at times i only see the cell and the stain at the later part of the images. so how do i conclude that this is colocalization of the cell? since i saw that in was within the cell before the imaging.
OK, first, colocalization:
A. If you can see the EGFP signal (green):
- The colocalization is between GFP (green) and anti-GFP (red), which, if it is overlap will give yellow signal.
- What you need to consider in this case would be the red, green and yellow in the merged picture. Do you always have red when you get green? In your case, it is possible that you will have red without green, but should not be the other way around. When you have yellow, you have colocalization. Depend on the intensity of yellow, the ratio between red, green and yellow signal in the merged pictures, you can say if there is significant or partial or no colocalization between red and green.
B. If the EGFP is too weak to see:
- you are trying to see if there is anti-GFP in cells.
- Do you always have red inside the cells? Or you have it outside the cells, too?
- When you do a merged picture of the full Z-stack (merge the full stak into one picture, and merge all 3 colour together), you can see if red is always inside the cells. If yes, then colocalization.
Second, Z stack:
- You see red in some slides but not others, that is expected, unless you expect that your GFP protein is distributed throughout the cells. Imagine your cells as a globe, with GFP protein concentrates at some places but not others. Of course when you scan the cells section by section, some will not have your protein, right. That is why you need to merge the z-stack, or do 3D projection, or do X-Z and Y-Z scan, or any combination of these method to show what you want to say.
if i understand you correctly you have a primary anyibody then a secondary with fluorochrome
the only thing you could colocalise with is the dapi and as this covers the entire nucleus that wont be too hard
i think what your saying is that the z series moves into the focal plane of the 647 then moves out of it again (again normal - sometimes, on bad days , the nuclear dye and your fluor are in different focal planes so you wont get them in the same z but thats quite rare as dapi and toto tend to cover most of the nucleus)
remember that a confocal is not giving you true 3d but moving around a central focal point also your antibody will appear different to a generic nuclear stain as it is more surface orientated
i think the answers in there some where but you questions a bit confusing
dom