Objective To explore the adaptability of clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) tissue transparency techniques in different organs of mice, and explore the compatibility of tissue transparency technology with nuclear staining and virus tracer neuron labeling technology. Methods （1） The brains, hearts and livers of freshly injected mice were extracted and dissected. Tissue blocks of 1.0cm×1.5cm×0.5cm were taken respectively, clear, unobstructed brain imaging cocktails and computational analysis-L (CUBIC-L) and clear, unobstructed brain imaging cocktails and computational analysis-R (CUBIC-R) tissue cleaning solution were used for transparency treatment. （2） The whole brain of mice was treated with transparency, and the nucleus of mouse brain neurons was stained with propidium iodide (PI). Three-dimensional nuclear volume images of the left and right hemispheres and cerebellar brainstem were obtained by light sheet fluorescence microscopy (LSFM). （3） Adeno-associated virus (AAV) -U6-enhanced green fluorescent protein (EGFP) was microinjected into the hippocampus and substania nigra of rat brain, respectively, to label neurons at specific anatomical sites. The rat brain after virus transfection was treated transparently. LSFM performs 3D stereoscopic imaging. Results The brain tissue, liver tissue and heart tissue all showed a transparent gel-like state after transparent treatment. However, the removal efficiency of different tissue blocks was different, and the removal efficiency of brain tissue was the highest, followed by liver tissue and heart tissue. LSFM could perform whole-volume three-dimensional imaging of transparent and nucleated brain tissue. AAV can clearly mark neurons in different parts, after transparent processing, the spatial distribution of these neurons could be observed, and the physical projection of neurons in different parts could also be observed. Conclusions CUBIC tissue transparency technology has universal application in brain tissue, heart tissue and liver tissue of mice, and the transparency efficiency of brain tissue is the highest. CUBIC tissue transparency method has good compatibility with nuclear staining and virus tracer neuron labeling technology.
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