Improved CLARITY Technology And Its Application In Liver Tissue Histology

Objective: To compare the application of four different concentrations of hydrogel in mouse brain,liver and kidney tissues,to explore the optimal ratio of hydrogel concentration in various tissue transparency processes,and secondly to optimize and improve CLARITY liver tissue hydrogel fixation transparency.Three-dimensional structure study of liver cancer model mice are based on this technology.Methods: 1.Prepare A4B4P4 according to the final concentration ratio of acrylamide(A),methylidene bisacrylamide(B)and paraformaldehyde(P)in the original hydrogel(A: B: P = 4%: 0.05%): 4%),A2B2P4(A: B: P = 2%: 0.025%:4%),A1B1P4(A: B: P = 1%: 0.0125%: 4%),A4B4P0(A: B: P = 4)%: 0.05%:0)hydrogel.Eight Kunming mice were randomly divided into 4 groups,2 in each group,which were perfused with A4B4P4,A4B4P0,A2B2P4,and A1B1P4 hydrogels,respectively,and then fixed and transparent.The relative transparency and protein loss of brain,liver and kidney tissues of mice treated with 4 kinds of hydrogels were calculated,and the brain,liver and kidney tissues were labeled with immunofluorescence to complete 3D reconstruction.2.Liver tissue fixation was performed using biphasic hydrogel.The fixed tissue was cut into 1 mm thick sections and randomly divided into two groups,SDS group and SDS+lipase group.SDS groups were cleared by sodium dodecyl sulfate for 9 days.The SDS+lipase group was firstly cleared with SDS for 5days,then immersed in lipase solution for 4 days.Both groups were subjected to transparent gray value determination and subjected to nuclear DAPI staining.Lastly,calculate the mark depth.3.Twenty-five SD rats were randomly divided into control group and model group.The model group was intraperitoneally injected with diethyl nitrosamine DEN for 18 weeks,and the control group was intraperitoneally injected with normal saline for 18 weeks.The liver tissues of the control group and the model group were stained with hematoxylin-eosin,and the liver tissues of the two groups were transparent,and then immunofluorescence labeling was performed to complete the three-dimensional reconstruction,and the average area of the lumen was measured.Results:1.The relative transparency of brain tissue in A2B2P4 group and A1B1P4 group was higher than that in A4B4P4 and A4B4P0 groups(P<0.05).The relative transparency of liver tissue in A1B1P4 group was higher than that in other three groups(P<0.05).The relative transparency of the kidney tissues of the A2B2P4 and A1B1P4 groups was higher than that of the A4B4P4 and A4B4P0 groups(both P<0.05).The protein loss of brain,liver and kidney in A4B4P0 group was higher than that in the other three groups(P<0.05).The protein loss in liver and kidney tissues of A2B2P4 group was lower than that in A1B1P4 group(P<0.05).2.During the passive lipid clearance process,the transparent gray value of liver tissue slices gradually increased from day 1 to day 5,but the liver tissue removal rate was slow afterwards,and the removal on day 10 was still opaque;On the7 th or 9th day,the relative transparent gray value of the two groups wascompared,the difference was statistically significant(P<0.01);the DAPI staining depth of the SDS+lipase group was 115 um,while the DAPI staining depth of the SDS group was 60 um,and the DAPI penetration of the SDS+lipase group The depth is 1.92 times that of the SDS group.3.The rat liver cancer model was successfully constructed.The pathological diagnosis was the liver cancer tissue structure.The tissue clearing technique was used to complete the immunofluorescence labeling of the control group and the model.The average area of the lobular motion and vein in the control group was lower than the average area of the liver cancer group(P <0.001).Conclusion: By comparing the application of four different concentrations of hydrogel in mouse brain,liver and kidney tissues,it is found that A2B2P4 and A1B1P4 hydrogels have better application in brain tissue transparency.A1B1P4 and A2B2P4 hydrogels are best application of liver tissue and kidney tissue respectively.By optimizing the CLARITY technology,it was found that the two-phase hydrogel tissue fixation combined with the lipase-immersed lipid passive removal technology can quickly realize the transparency of mouse liver tissue.Based on the liver cancer model mice,the CLARITY technique was used to reconstruct the three-dimensional structure of liver cancer tissues,and the distribution of spatial positional relationship in the three-dimensional structure of the tissues was realized,which promoted the pathological study of liver in CLARITY technology.