Characterization of the cellular and subcellular distribution of fms-like tyrosine kinase 3 (FLT3) and other neuronal proteins using an alkaline phosphatase (AP) immunolabeling method

Precisely localizing the spatial distribution of proteins within various cell types in the brain, and in sub-cellular compartments such as the synapses, is critical for generating and testing hypothesis to elucidating the function of target proteins in the brain. The fms-like tyrosine kinase-3 (FLT3) has been studied extensively in the context of blood cell development and leukemia pathogenesis, but little is known about its role in the brain. Previous work characterizing FLT3 protein expression in the brain have not yielded convincing results, mainly limited by the low expression level of FLT3 and poor sensitivity of standard immunolabeling method using fluorescent secondary antibody. In this study, we report the systematic characterization of FLT3 protein localization during brain development using a highly sensitive immunolabeling methodology based on alkaline phosphatase (AP) polymer biochemistry. Our results reveal a previously unknown neuron-selective FLT3 expression pattern in both mouse and human cerebellum tissue samples, and demonstrate a gradual increase in the total FLT3 protein level and a cytosolic-to-dendritic change in subcellular FLT3 distribution during mouse cerebellum development. Through combining the AP immunolabeling of FLT3 with standard immunostaining of various cell type markers to achieve hybrid co-labeling of multiple antigens in tissue sections, we demonstrate that the main cell type that express FLT3 in the cerebellum is PV+, Calbindin+ Purkinje cells. To expand the use case of AP immunolabeling method in labeling neuronal proteins, we show robust labeling of Kir2.1, a potassium channel protein that is expressed at low level in neurons, in brain tissue sections collected from mouse, pig, and human brains. We further established that the AP immunolabeling method can be used in human stem cell-derived neurons to detect postsynaptic density scaffold protein PSD95 within fine subcellular structures such as dendritic spines and synapses, in cultured primary mouse cortical neurons and human stem cell-derived neurons. To our knowledge, our work represents the first report of applying AP immunolabeling to detect neuronal protein expression in brain tissue and cell samples. Moreover, our work reveals a previously unknown neuron-specific pattern of FLT3 expression in the brain, providing the foundation for further mechanistic studies to uncover its role in brain development and functioning.