Quantification of immunohistochemistry depends on a number of parameters including what the distribution of the staining looks like for each antigen. Is it somal (cell body), neuritic, puntca-like, etc? Depending on this you have many choices for quantification. Are you looking to see if all three labels are in the same cell or same field, if their intensities vary, etc? The choice of how one quantifies is dictated by what the staining looks like, and what you are trying to achieve. I've used ImageJ, Imaris, and in-house methods to quantify- each used for very specific types of staining. I also cannot stress enough that your staining needs to as good as you can possibly get for accurate measures.  We probably need more information to be helpful to you and  why Image J wasn't accurate enough.

Julie Lauterborn

Dear Dr.Lauterborn 

I am trying to quantify the florescence intensity of different dextrans to asses the blood brain barrier permeability. I have several brain sections and I did the staining only for the capillaries ( blue) and two different dextrans (red and green) already florescence tagged .

All I need to quantify is the amount of each dextran inside the brain since I removed all the blood from the capillaries after the injection. 

Is the dextran labeling diffuse?  Without seeing what one of your images looks like it is difficult to tell you how to try to quantify. But, if diffuse, you could try sampling for intensity levels across the images (taking measures in different places but for each measure you would have the red and green integrated densities, and then look at the correlations across all of your samples) or look at the maximal, and median intensity levels across the image for each dextran if they are not equally dispersed within the image. Does it look like you have a relatively equal diffuse of both dextrans? A potential problem that you may want to consider if you are trying to compare the two dextrans, is whether your green and red are being picked up equally by your camera, etc so that you are not optimizing one over the other by just how you are imaging; one way to check this is to flip the tags for each.

Dr.Lauterborn you just asked most of the questions I was thinking about while doing the imaging.

First yes the dextran labeling diffuse and I can send you some images if you don't mind.

Second yes I am trying as much as possible to overcome the problem with the microscope for picking the green and red specifically.

I will show you some of the images and I really appreciate your help.

Thank you so much