Immunocytochemical detection of two nuclear proteins within the same neuron using light microscopy

We developed a method of double immunocytochemistry (ICC) that can be used with conventional light microscopy for localizing two different nuclear proteins. The procedure involves two sequential rounds of ICC that both employ the avidin and biotin conjugated enzyme (ABC) amplification method, separated by an Avidin D and biotin blocking step to reduce non-specific avidin-biotin reactions. Round one of ICC employs the use of avidin and biotin conjugated alkaline phosphatase (ABC-AP) and the Vector Red (VR) substrate, which produces a red colorimetric reaction product. The second round of ICC makes use of avidin and biotin conjugated peroxidase (ABC-HRP) and the Vector(R) SG substrate, which produces a gray colorimetric reaction product. Neuronal nuclei that are double-labeled for both proteins appear red with a gray core. This protocol allows the simultaneous detection of two proteins within the same subcellular compartment of a single neuron, without the need for epifluorescence or scanning confocal laser microscopy.