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Quanitication with Image J for fluorescence in endothelial monolayers - (Jan/25/2011 )

Hello all and thank you for taking the time to consider this problem.

I am working with Bovine Aortic Endothelial Cells and have found a treatment which seems to increase convective water flux in monolayers grown on filters. I would like to quantify this expected protein expression change via cytochemistry utilizing Alexia-488 and primary antibody for aquaporin-1. I am currently working with Image J and I think I want to use a threshold determined from my controls to obtain protein expression quantification. If I can do this I can contrast the difference between treated and untreated samples. My problems are finding a robust methodology which can be applied to monolayers systematically. Ideally the program would threshold out the noise (from control with secondary Ab only) and then over the area of fluorescence provide a value representing AQP-1 expression for the entire monolayer. I could then run this on all my treated monolayers to make conclusions on the effect of the treatment even with differing monolayer formation between samples.

Could anyone provide a good guide to doing this using image j and matlab?


Also I am using confocal microscopy to obtain images in z stacks.