I was wonder if anyone has experienced this problem. I am performing immunofluorescence with smooth muscle 22 alpha (Abcam ab10135) on mouse embryonic tissue E13.5. The tissue is fixed using methanol and DMSO (4:1) then paraffin embedded. Sections are 5um thick. Concentration of my primary antibody is 5ug/ml, while the secondary Donkey anti-rabbit Alexa555 is at 10ug/ml. My problem is that I see the localization of specific signal change within the slide. For example, one section will display the correct cytoplasmic expression of smooth muscle 22 alpha in the ascending aorta, but in an adjacent section on the same slide the cytoplasmic expression is gone and expression is only seen within the nuclear membrane. The tissue sections are on Superforst plus slides. Could this be a reason for the changes in expression.?