. Cell-level analysis and a growing appreciation of cell-type diversity have transformed our understanding of the brain. Beyond first-order classification of cells as glial or neuronal, excitatory or inhibitory, we now know there are dozens of molecularly-defined cell-types that differ in their morphology, connectivity, physiology, and gene & protein expression. Detection of protein in fixed tissues via immunohistochemistry (IHC) has been a major driver of cell-type discovery, with a cell?s precise microenvironment within tissue (e.g., proximity to vasculature) further defining its identity. Such data have yielded a rich picture of how cellular topography varies by brain region and provides a robust backdrop to assess cell-type -specific changes that occur in disease states, such as loss of parvalbumin-expressing inhibitory interneurons in postmortem brains from schizophrenia patients. Despite the prevalence of IHC its application has remained encumbered by the slow rate at which reagents such as IgG antibodies passively diffuse into tissue. Due to this bottleneck, tissues have traditionally been thinly sliced (? 50 m) to facilitate uniform staining of their features and make quantitative analysis reliable. CLARITY, iDISCO, and related techniques that optically-clear intact tissues by removing cell membrane lipids have offered a means to perform whole-brain IHC, as delipidation grants reagents easier access to deep tissue sites. However, labeling time remains a major bottleneck, with intact samples requiring weeks to months of incubation for labeling to reach the center. If whole organs could be labeled more quickly and practically it would create a powerful tool, such as for performing unbiased molecular- phenotyping of models of developmental disorders such as autism thought to involve subtle changes in rare cell-types. To this end we have developed SmartLabel (SL), the world?s first whole-organ active immunostaining device that can fully label an entire mouse brain in just 1-3 days using a form of electrophoresis. SL makes use of SWITCH, a method in which antibodies are evenly distributed throughout the tissue before binding to target proteins is ?switched on,? to produce labeling that is strikingly uniform in intensity from the sample?s surface to its core. To broaden SL?s range of applications, we propose to: (1) extend SL?s rapid immunolabeling capability from tissues processed using CLARITY to those prepared using iDISCO, and ensure compatibility with key morphological, cell-type, and neuronal activity markers such as cFos. We will also adapt the technology to work with diverse sample types such as cerebral organoids, a patient-derived in vitro model of clinical tissue samples. (2) We will prototype and test a next-generation SL that enables cost- effective cohort-level immunolabeling of 20 organoids or 8 adult mouse brains simultaneously, a 4-fold increase from the current design. Upon completion of this Phase 1 project we will have developed a rapid immunostaining device capable of turnkey batch-processing of a wide range of sample types, facilitating application of quantitative whole-sample analysis techniques in neuroscience and related biomedical fields.
. The ability to reveal the molecular identity and anatomical microenvironment of each cell in organs such as the brain would enable unbiased screens of human disease models and offer insight into disorders like schizophrenia and autism that are thought to involve subtle changes in rare cell-types. LifeCanvas Technologies? SmartLabel provides for rapid (1-3 day) labeling of intact rodent organs with antibodies and other charged molecular probes, an order of magnitude faster than traditional passive labeling and with staining intensity that is uniform from the sample?s surface to its center. By making SmartLabel compatible with a broader range of samples and establishing turnkey batch-processing capability, we will enable researchers to perform quantitative whole-organ molecular-phenotyping studies and investigate whether specific cell-types and the circuits they form are aberrant in disease and addressable by therapeutic intervention.
