To visualize the structure and organization of the brain is a fundamental requirement in the research of neuroscience. Here, combining with two-photon excitation fluorescence microscopy and transgenetic mouse GAD67, we demonstrate a custom-built second harmonic generation (SHG) microscope to discriminate brain layers and sub regions in the cerebellum and brain stem slices with cellular resolution. In particular, the cell densities of neurons in different brain layers are extracted due to the cell soma appearing as dark shadow on an SHG image. Further, the axon initial segments of the Purkinje cell are easily recognized without labeling, which would be useful for guiding micropipettes for electrophysiology.