{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T06:00:39Z","timestamp":1792130439040,"version":"4.4.0"},"reference-count":21,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2025,3,4]],"date-time":"2025-03-04T00:00:00Z","timestamp":1741046400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"SFARI Bridge to Independence","award":["NIH R01NS138758"],"award-info":[{"award-number":["NIH R01NS138758"]}]},{"name":"SFARI Bridge to Independence","award":["NIH R21EY033550"],"award-info":[{"award-number":["NIH R21EY033550"]}]},{"name":"SFARI Bridge to Independence","award":["#S10OD030322"],"award-info":[{"award-number":["#S10OD030322"]}]},{"name":"Harvard Stem Cell Institute fund","award":["NIH R01NS138758"],"award-info":[{"award-number":["NIH R01NS138758"]}]},{"name":"Harvard Stem Cell Institute fund","award":["NIH R21EY033550"],"award-info":[{"award-number":["NIH R21EY033550"]}]},{"name":"Harvard Stem Cell Institute fund","award":["#S10OD030322"],"award-info":[{"award-number":["#S10OD030322"]}]},{"name":"IDDRC Cellular Imaging Core","award":["NIH R01NS138758"],"award-info":[{"award-number":["NIH R01NS138758"]}]},{"name":"IDDRC Cellular Imaging Core","award":["NIH R21EY033550"],"award-info":[{"award-number":["NIH R21EY033550"]}]},{"name":"IDDRC Cellular Imaging Core","award":["#S10OD030322"],"award-info":[{"award-number":["#S10OD030322"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Precisely localizing the spatial distribution of proteins within various brain cell types and subcellular compartments, such as the synapses, is essential for generating and testing hypotheses to elucidate their roles in brain function. While the fms-like tyrosine kinase-3 (Flt3) has been extensively studied in the context of blood cell development and leukemia pathogenesis, its role in the brain remains poorly understood. Previous efforts to address this issue were hindered by the low expression levels of Flt3 and the limited sensitivity of the standard immunolabeling method, which were insufficient to reliably detect Flt3 protein in brain tissue. In this study, we systematically characterized Flt3 protein localization during brain development using a highly sensitive immunolabeling method based on alkaline phosphatase (AP) polymer biochemistry. This approach revealed a previously unrecognized neuron-selective Flt3 expression pattern in both mouse and human cerebella, with a developmental increase in total protein levels accompanied by a shift from a cytosolic to a dendritic subcellular distribution. Combining AP-polymer-based immunohistochemistry (AP-IHC) for Flt3 with conventional immunostaining of cell type marker proteins revealed parvalbumin- and calbindin-positive Purkinje cells to be the main cell type expressing Flt3 in the cerebellum. To validate the versatility of the AP-IHC method for detecting low-abundance neuronal proteins, we demonstrated robust labeling of Kir2.1, a potassium channel protein, in brain tissue sections from mouse, pig, and human samples. We further applied the AP-IHC method to human stem cell-derived neurons, effectively visualizing the postsynaptic density scaffold protein PSD95 within synapses. To our knowledge, this is the first study to employ an AP-IHC method combined with other standard immunofluorescent staining to co-detect weakly expressed neuronal proteins and other cellular markers in brain tissue and cultured neurons. Additionally, our findings uncover a previously unrecognized neuron-specific pattern of Flt3 expression in the cerebellum, laying the foundation for future mechanistic studies on its role in normal brain development and neurological disorders.<\/jats:p>","DOI":"10.3390\/ijms26052284","type":"journal-article","created":{"date-parts":[[2025,3,4]],"date-time":"2025-03-04T07:47:37Z","timestamp":1741074457000},"page":"2284","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Visualizing the Cellular and Subcellular Distribution of Fms-like Tyrosine Kinase 3 (Flt3) and Other Neuronal Proteins Using Alkaline Phosphatase (AP) Immunolabeling"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0980-6954","authenticated-orcid":false,"given":"Yuqin","family":"Yin","sequence":"first","affiliation":[{"name":"Department of Neurosurgery, Boston Children\u2019s Hospital, 300 Longwood Ave, Boston, MA 02115, USA"},{"name":"F. M. Kirby Neurobiology Center, Boston Children\u2019s Hospital, 3 Blackfan Circle, Boston, MA 02115, USA"},{"name":"Department of Neurosurgery, Harvard Medical School, Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kathleen Z.","family":"He","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery, Boston Children\u2019s Hospital, 300 Longwood Ave, Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jane","family":"Kirby","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery, Boston Children\u2019s Hospital, 300 Longwood Ave, Boston, MA 02115, USA"},{"name":"F. M. Kirby Neurobiology Center, Boston Children\u2019s Hospital, 3 Blackfan Circle, Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ishraq A.","family":"Haque","sequence":"additional","affiliation":[{"name":"Harvard College, Cambridge, MA 02138, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2883-2610","authenticated-orcid":false,"given":"Xin","family":"Tang","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery, Boston Children\u2019s Hospital, 300 Longwood Ave, Boston, MA 02115, USA"},{"name":"F. M. Kirby Neurobiology Center, Boston Children\u2019s Hospital, 3 Blackfan Circle, Boston, MA 02115, USA"},{"name":"Department of Neurosurgery, Harvard Medical School, Boston, MA 02115, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"eaay5947","DOI":"10.1126\/science.aay5947","article-title":"An atlas of the protein-coding genes in the human, pig, and mouse brain","volume":"367","author":"Sjostedt","year":"2020","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1433","DOI":"10.1152\/physrev.00029.2018","article-title":"FMS-like Tyrosine Kinase 3\/FLT3: From Basic Science to Clinical Implications","volume":"99","author":"Kazi","year":"2019","journal-title":"Physiol. 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