Fig. 5. Enhanced thrombosis reduced spontaneous bleeding and secondary hemophilia complication.
(A) Paraffin-embedded liver tissues were prepared without perfusion to prevent the loss of evidence of spontaneous bleeding (WT, n = 3; F8I22I, F8I22I-LNP, F9Mut, and F9Mut-LNP, n = 4). RBCs in the interstitial liver tissue [marked by black triangles in the liver tissue using hematoxylin and eosin (H&E) staining] were measured by immunofluorescence staining by detecting the autofluorescence signal at 470 and 540 nm. The intensity of the coexpressions (yellow signals) were then calculated. Ten areas of the liver were randomly selected from each mouse, and each measured autofluorescence signal was subjected to the RBC frequency analysis. Each dot indicates a signal from one selected area. Correlation analysis was conducted using the mean yellow fluorescence values obtained from each mouse. Blue dot, LNP-CRISPR-mAT groups; red dot, control group. Scale bars, 100 μm. (B) The whole kidney was formalin fixed, and gross histology was examined using H&E staining. The number of abnormally shaped glomerular capsules (black triangles) was calculated from three randomly selected regions of the cortex (1 mm2) per mouse and analyzed. **P < 0.01 and ***P < 0.001.