Triple immunofluorescent study of biocytin-filled neuron. A. Biocytin-filled neuron was visualized by rhodamine red-X-conjugated streptavidin. B and C showed ChAT- and nestin-immunoreactive neurons visualized by cy2 (blue) and alexa 405 (green) respectively. D. Image merged from A, B and C. The red arrow pointed to the cell double labelled by the nestin and ChAT antibodies and filled with biocytin by whole-cell patch clamp recording. The white arrow pointed to the cell double labelled by ChAT and nestin antibodies. The blue arrow pointed to a neuron that only expressed ChAT. Because the brain slice was made from neonatal rat and did not perfuse transcardially before slice preparation, there were some epithelium lining blood vessels labelled by nestin monoclone antibody in C and D. Scale bar was 20 μm.