Plastic Embedding Method For Multiple Fluorescent Labeled Samples

Revealing the fine structure of neural circuit is important for understanding brain function.Fluorescence Micro-optical Sectioning Tomography(f MOST)is one of the representative techniques for obtaining high-quality and high-resolution brain-wide data.The establishment of the whole brain plastic embedding method that preserves multiple fine structural information while maintaining good cutting and imaging performance is of great significance to the development of this technology.This paper focuses on the unresolved issues in the preparation of whole brain samples,such as poorly maintained contours of the whole-tissue,unstable continuous cutting with micron thickness,and low fluorescence preservation rate of red fluorescent protein and chemical dye/probe.Therefore,based on the above questions,with the aim of solving the scientific problems of multi-structure information acquisition of neural circuits,the following work is completed.(1)We established a stable plastic embedding method(GMA-S)for whole brain samples with lower background,that can be prepared in mass.We reduced the changes in the morphological structure of the mouse brain by standardizing the mouse perfusion process;by optimizing the resin composition,the hardness and hydrophobicity of the embedded sample were improved,enabling continuous and stable ultra-thin sectioning.At the same time,combined with real-time dyeing of nucleic acid dyes,a standardization strategy for whole brain plastic embedding was established,GMA-S,which can be used for preparation of large numbers of samples stably.GMA-S embedding method can maintain the shape of the whole-brain and fine structure of GFP labeled samples.The embedded samples have good hardness and hydrophobicity to meet the requirements of long time micron cutting,and can obtain the cytoarchitecture staining information during whole-brain imaging.With the combination of embedding and f MOST imaging,we acquire the brain-wide data set of cholinergic and pyramidal neurons with cytoarchitecture reference information.We analyzed the distribution of cholinergic neurons,the projection pattern of cholinergic neurons in motor nuclei,the dendritic morphology and axon long-range projection pattern of Thy1 neurons in cortex.(2)We established a plastic embedding method(GMA-D)for preservation of multicolor fluorescent protein.The red fluorescent protein(RFP)has a low fluorescence preservation rate and a high auto-fluorescence in red channel after plastic embedding.The organic reagent and higher polymerization temperature during the plastic embedding process affect the structure of the red fluorescent protein,and lead to a decrease the fluorescence preservation and increase in auto-fluorescence of the tissue.In this paper,the addition of a fluorescent protective agent increased the red fluorescent protein preservation rate,reduced the polymerization temperature by replacing the initiator.We developed an embedding method for red fluorescent protein,GMA-D,and this method is also suitable for GFP and BFP.GMA-D embedding method nearly doubled the retention of red fluorescent protein.With the combination of embedding and the f MOST imaging,we obtain the brain-wide data set of VIP neurons in transgenic mice.The distribution and morphological information of VIP neurons in the cortex were reconstructed and analyzed.Based on the simultaneous labeling of two fluorescent proteins,we studied the projection and input patterns of cholinergic nucleus in the pons on a single sample,and find that cholinergic neurons receive projections of cholinergic neurons from other brain regions.(3)We studied the main factors that affect the fluorescence preservation of dyes/probes fluorescence preservation,and developed a plastic embedding method(GMA-T)for preservation of multicolor fluorescent dyes/probes.Chemical fluorescent dyes/probes have the advantages of diversity and flexibility,been widely used in research in neuroscience to label multiple structures.Plastic embedding can reduce the fluorescence intensity of the chemical fluorescent dyes/probes.Here,we combined the new background inhibitor(TB)with whole-brain plastic embedding,optimized the concentration of TB used in plastic embedding,and established an embedding method(GMA-T)for chemical fluorescent dyes/probes to meet the needs of simultaneous acquisition of multiple structural information.GMA-T embedding method can also maintain the fine structure of the commonly used chemical fluorescent dyes/probes and fluorescent protein.Combined with the whole brain optimized imaging system,multiple structural information of neural system structure was preliminary obtained.In this study,a variety of whole-brain plastic embedding methods were established and optimized,for multiple fluorescent labeled samples to meet the requirements of simultaneous acquisition of multiple fine structures.The improved approach improve the imaging quality of fine structure of neural circuits.Therefore,we propose that the whole brain plastic embedding methods established here combined with f MOST imaging is a routine tool for study on neuroscience,such as neural circuit and neuronal morphological features at the single cell level.