A cost- and time-efficient method for high-throughput cryoprocessing and tissue analysis using multiplexed tissue molds.

Cryosectioning remains the gold standard for antibody and transcriptomic/in situ hybridization tissue analysis. However, tissue processing is time-consuming and costly, limiting routine and diagnostic use. Currently, no commercially available protocols or products exist for multiplexing this process. Here, we introduce multiplexed tissue molds (MTMs) that enable high-throughput cryoprocessing-cutting costs and workload by up to 96% while permitting the processing of tissues of various sizes and origins. We demonstrate compatibility with heterogeneous tissues by processing 19 different adult mouse tissues in parallel. Furthermore, we process up to ∼110 neural organoids of different ages and sizes simultaneously and assess their neural differentiation marker expression. MTMs allow sectioning-based tissue analysis when labor, time, and cost are limiting factors. MTMs could be used to compare high specimen numbers in histopathological settings, organism-wide antigen and antibody targeting studies, high-throughput tissue screens, and defined tissue section positioning for, e.g., spatial transcriptomics experiments.