Fig. 5

Large-volume imaging of AKS-cleared tissue using two-photon microscopy imaging. A, B 3D imaging of 150-μm-thick slice from Thy1-YFP mice before and after AKS treatment. A XZ plane showing YFP signal at different imaging depths before and after AKS treatment. B XY plane of YFP signal at different imaging depths before and after AKS treatment. Representative of n = 3 slices each conditioning (scale bar: A, 50 μm; B, 200 μm). C–F Imaging of the cortex of AKS-cleared hemibrain of Thy1-YFP mice. Representative of n = 2 hemibrain. C The imaging diagram. D 3D reconstruction of YFP-labelled neurons in the cortex (scale bars: 300 μm). E Top view of the 3D reconstruction (scale bars: 300 μm). F Optical sections of AKS-cleared Thy1-YFP mouse brain at various depths (scale bars: 200 μm). G–K 3D imaging and analysis of the tdTomato-labelled CRH neurons in CRH-cre;Ai14 mice PVN. Representative of n = 2 slices. G Distribution of CRH neurons in different locations of the hypothalamus (scale bars: 100 μm). H Top view of 3D reconstructed CRH neurons in PVN (scale bars: 100 μm). I Side view of the 3D reconstructed CRH neurons in PVN (scale bars: 100 μm). J, K Reconstruction of the surface of the PVN area (J) and the location of CRH neurons (K) in PVN (scale bar: 100 μm). L Reconstructed 3D distribution of CRH neurons in PVN (scale bar: 50 μm)