Transgenic mice (Trangenic line M)which express GFP in cortical-layer-5 pyramidal neurons were surgically treated to expose the dura. An optical chamber was constructed over the dura using agarose (Sigma A9793)and custom-made cover glass, sealed with dental acrylic.A titanium bar with screw holes was embedded into the acrylic so the animal could by stablized during imaging sessions.
Imaging of neurons expressing GFP was obtained using custom image-acquisition software (MatLab) and Zeiss microscope.
Immunocytochemistry was performed on 60um vibratome sections using primary antibody rabbit anti-GFP (Chemicon AB3080) with secondary antibody biotinylated goat anti-rabbit (F)ab fragment (Jackson Labs) and ABC method of staining (Vector Labs) with DAB.
Electron Microscopy was used to obtain a three-dimensional ultastructure of dendrites.