CLARITY Constructs Liver Translucency Model And Related Applications

Objective:To explore a rapid hepatic tissue translucency technique based on the CLARITY,optimize the animal model of liver cirrhosis by three-dimensional immunofluorescence labeling with condition optimization,and develop a paraffin section antibody elution technique based on this technique.Methods:(1)The rats were perfused with hydrogel,the liver was im mobilized by hydrogel and cut into 1mm slices.The slices were randomly divided into two groups.The control group was treated with conventional passive lipid removal.The experimental group was placed in a special electrophoresis device.The lipids were washed under external electric field conditions.The optimal liver transparence was explored by increasing the electrophoresis temperature and quantified by relative transparency.(2)DEN-induced rat liver cirrhosi tissues were cleared by optimized liver clearing conditions Immunofluorescence was used to explore imaging conditions in various rat organ tissues,and then,confocal microscope was performed on rat liver cirrhosis tissues imaging.(3)Combining CLARITY with paraffin sections,which has propertiesof hydrogel fixation,lipid removal,and antibody elution,to explore the o ptimal conditions for the transparency method of paraffin sections and to compare with conventional acid elution methods.The antibody elution effect was determined by comparing the change in integral fluorescence before and after elution of the immunofluorescent labeling section.Results:(1)Under the condition of 12 V 37?,the experimental group become transparent after 48 h electrophoresis,the relative-transparency is changeless when run with another 48 h electrophoresis.If elevate the temperature of electrophoresis,after 48 h basal electrophoresis(12V 37?),the relative-transparency can be further improved.(2)The spatial distribution of tissue structures(basal membrane and astrocytes)in rat kidney and brain was successfully marked by tissue translucency treatment.Rat liver cirrhosis proved by pathological examination,laser confocal scanning three-dimensional imaging successfully demons trated the distribution and relationship of the spatial structure of the blood vessels,pseudolobules and hepatocytes in the cleared cirrhotic tissues.(3)Using 4um tissue sections,hydrogel fixation,and cationic detachment slides can enhance the resistance of paraffin sections to elution.The paraffin section processed by the transparent step can be used to obtain the best antibody elution effect using 4% SDS at 60?.The effect is sign ificantly better than the conventional acid elution method.That is,the ave rage integral fluorescence intensity of the slice decreases most after elution,and The counterstaining effect is good.Conclusion:To create extremely clear liver tissue,the best electroph oretic tissue clearing procedure is to run the samples at 12 V 37? for 48 h followed by more 48 h with the temperature increased to 52?.Liver cirrhosis tissues were treated with hepatic rapid clearing method.The position and relationship of high-resolution fine structures in cirrhotic tissue in three-dimensional space were visualized by confocal laser scanning microscopy.CLARITY is in good agreement with paraffin sections.Transpare nt sectioning of paraffin sections enables immunofluorescent staining of the sections followed by repeated antibody-eluting and counterstaining,which protects the morphological structure and antigen-binding sites of the tissue from occurring change.