Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Factor XI localization in human deep venous thrombus and function of activated factor XI on venous thrombus formation and hemostasis

doi: 10.1016/j.rpth.2025.102720

Figure Lengend Snippet: Presence of factor (F)XI in human deep vein thrombosis (DVT). (A) Representative immunofluorescent images of fresh components of DVT. The upper row shows CF488-labeled FXI (green), CF568-labeled fibrin (red), and merged images. The second row shows CF488-labeled FXI, CF568-labeled glycophorin A, and merged images. The third row shows CF488-labeled FXI, CF568-labeled CD42b, and merged images. The lower row shows CF488-labeled FXI, CF568-labeled CD66b, and merged images. FXI was closely localized with fibrin. Glycophorin A, a marker of erythrocytes, was predominantly surrounded by FXI. FXI was partly localized with CD42b, a marker of platelets, but hardly localized with CD66b, a marker of neutrophils. (B) Representative immunofluorescent images of fresh components of DVT show CF488-labeled FXI, CF568-labeled FXII, 4,6-diamidino-2-phenylindole (DAPI, a DNA marker), and merged images. FXI was partly localized with DNA and FXII. (C) Representative images of in situ nanogold labeling and electron microscopy of fresh components of DVT. The left upper image shows an immunohistochemical image of FXI. Nanogolds accumulate at the 3, 3′-diaminobenzidine tetrahydrochloride deposition site against anti-FXI antibody (left lower). Nanogold deposition was localized on a fibrin-like mesh network but not on the neutrophils (arrow). (D) Representative immunohistochemical images in fresh and organizing areas of DVT. The organizing areas were confirmed by the presence of CD34-immunopositive cells, corresponding to endothelialization/organization, while the fresh area was confirmed by the absence of CD34-immunopositive cells. FXI localized fibrin- and erythrocyte-rich fresh areas. In the fresh area, aggregated clusters of platelets and sparsely distributed neutrophils are present. The inset in CD66b represents a high-magnification image of the dashed square. In the organizing area, immunoreaction for FXI, fibrin, glycoprotein (GP) IIb/IIIa, glycophorin A, or CD66b is modest or focal. (E) Immunopositive area for FXI, fibrin, GPIIb/IIIa, and glycophorin A, and CD66b-immunopositive cell density in nonorganizing (CD34-immunonegative) and organizing (CD34-immunopositive) areas (Mann–Whitney U-test). HE, hematoxylin and eosin.

Article Snippet: CF488 conjugated donkey anti-sheep immunoglobulin (Ig) G (Biotium) and CF568 conjugated donkey anti-rabbit IgG (Biotium) or CF568 conjugated donkey anti-mouse IgG (Biotium) were used as secondary antibodies. shows an immunopositive band for human FXI (HFXI 1111, Enzyme Research Laboratories Ltd), but not human prekallikrein (HPK 1302, Enzyme Research Laboratories Ltd), in Western blotting using the anti-FXI antibody (LS-B10243; LSBio).

Techniques: Labeling, Marker, In Situ, Electron Microscopy, Immunohistochemical staining, MANN-WHITNEY

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Factor XI localization in human deep venous thrombus and function of activated factor XI on venous thrombus formation and hemostasis

doi: 10.1016/j.rpth.2025.102720

Figure Lengend Snippet: Effects of ONO-1600586 on human activated factor (F)XI (FXIa) and in vitro thrombus formation. The human blood was perfused in a flow chamber under a low-shear rate of 70/s. (A) Representative fluorescent images under a low-shear rate (70/s). Platelets and leukocytes were labeled with mepacrine, and the fluorescent images were captured 3, 6, and 9 minutes after perfusion. The surface covering area time-dependently increased in all 3 groups. Large fluorescent dots indicate leukocytes (arrows). (B) The surface covering areas under a low-shear rate in the control (solvent), ONO-1600586, and rivaroxaban addition (2-way repeated measure anova with Tukey’s multiple comparisons test). (C) Representative immunostaining images of fibrin on the glass coverslips after perfusion. ONO-1600586 or rivaroxaban addition decreased fibrin formation. (D) Fibrin-immunopositive area after human blood perfusion under a low-shear rate (Kruskal–Wallis test with Dunn’s multiple comparison test). (E) The number of leukocyte adhesions after human blood perfusion under a low-shear rate (Kruskal–Wallis test). (F) Activated FX activity, FXIa activity, and levels of prothrombin fragment 1 + 2 before and after perfusion of human blood (2-way repeated measure anova with Tukey’s multiple comparison test). ∗ P < .0001 vs control; † P < .0001 vs ONO-1600586; ‡ P < .01 vs ONO-1600586. (G) Representative immunofluorescent images of the glass coverslips after perfusion. The upper row shows CF488-labeled FXI (green), CF568-labeled fibrin (red), and merged images. The second row shows CF488-labeled FXI, CD42b, and merged images. The lower row shows CF488-labeled FXI, CF568-labeled CD66b, and merged images. FXI was closely localized with fibrin and partly around CD42b, a marker of platelets, but hardly localized with CD66b, a marker of neutrophils.

Article Snippet: CF488 conjugated donkey anti-sheep immunoglobulin (Ig) G (Biotium) and CF568 conjugated donkey anti-rabbit IgG (Biotium) or CF568 conjugated donkey anti-mouse IgG (Biotium) were used as secondary antibodies. shows an immunopositive band for human FXI (HFXI 1111, Enzyme Research Laboratories Ltd), but not human prekallikrein (HPK 1302, Enzyme Research Laboratories Ltd), in Western blotting using the anti-FXI antibody (LS-B10243; LSBio).

Techniques: In Vitro, Shear, Labeling, Control, Solvent, Immunostaining, Comparison, Activity Assay, Marker

Journal: eLife

Article Title: High-throughput expansion microscopy enables scalable super-resolution imaging

doi: 10.7554/eLife.96025

Figure Lengend Snippet:

Article Snippet: CF568 Donkey Anti-Rabbit IgG (H+L) , Biotium , 20098 , 1:500.

Techniques: Concentration Assay

Journal: Nature Communications

Article Title: Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles

doi: 10.1038/s41467-024-48991-x

Figure Lengend Snippet: a Scheme of SARS-CoV-2 genome with constructs used for its detection in infected cells. 48 antisense DNA oligonucleotide probes were used to target the ORF1a-coding region of vgRNA that is exclusive to the positive-sense vgRNA and does not occur in the sgRNAs. The RNA FISH probes are conjugated with AF647 or CF568. b Representative confocal images of vgRNA in infected Vero E6 cells at 6 hpi display scattered diffraction-limited (DL) puncta. c Representative SR image of an infected cell at 6 hpi reveals distinct vgRNA clusters in the cytoplasm. d Zoomed-in region of the SR image (green frame in c ) displays an agglomeration of vgRNA clusters. e Zoomed-in region of the SR image (red frame in c ) shows nanoscale puncta of individual vgRNA molecules. f Representative confocal images of vgRNA in infected Vero E6 cells at 24 hpi display large DL foci in the perinuclear region of the cytoplasm. g Representative SR image of an infected cell at 24 hpi reveals large perinuclear vgRNA clusters. h Zoomed-in region of the SR image (blue frame in g ) displays dense vgRNA clusters. i Zoomed-in region of the SR image (yellow frame in g ) displays nanoscale puncta of vgRNA molecules. j BIC-GMM cluster analysis of the region shown in d . k BIC-GMM cluster analysis of the cell shown in g . l BIC-GMM cluster analysis of the region shown in h . m Histogram of the radii of gyration (Rg) of the vgRNA clusters indicate their size increase between 6 hpi (tan) and 24 hpi (blue). Data for the histograms are obtained from 10 cells (6 hpi) and 16 cells (24 hpi) and 2 independent experiments for each time point. Scale bars, 10 µm ( b , f ), 2 µm ( c , g , k ), 500 nm ( d , e , h , i , j , l ). Dashed lines in c and g indicate the boundary of the cell nucleus (large dark region). Localizations that belong to the same cluster in j , k , l are depicted with the same color, but colors are reused. Color bars in c – e , g – i show the number of single-molecule localizations within each SR pixel (20 × 20 nm 2 ).

Article Snippet: Secondary antibodies and the optimal dilutions and concentrations used are as follows: AF647-conjugated donkey anti-mouse IgG (Thermo Fisher, A-31571, lot 2555690, 1:500, 4 µg/mL), AF647-conjugated donkey anti-rabbit IgG (Thermo Fisher, A-31573, lot 2359136, 1:500, 4 µg/mL), AF647-conjugated donkey anti-sheep IgG (Thermo Fisher, A-21448, lot 2454134, 1:500, 4 µg/mL), CF568-conjugated donkey anti-goat IgG (Sigma-Aldrich, SAB4600074-50UL, lot 18C0723, 1:500, 4 µg/mL), CF568-conjugated donkey anti-rabbit IgG (Sigma-Aldrich, SAB4600076-50UL, lot 21C1025, 1:500, 4 µg/mL), CF568-conjugated donkey anti-mouse IgG (Sigma-Aldrich, SAB4600075-50UL, lot 17C1116, 1:500, 4 µg/mL), CF568-conjugated donkey anti-sheep IgG (Sigma-Aldrich, SAB4600078-50UL, lot 11C1031, 1:500, 4 µg/mL), CF583R-conjugated donkey anti-mouse IgG (Biotium, Custom CF Dye, lot 23C1122, 1:250, 4 μg/mL), CF583R-conjugated donkey anti-rabbit IgG (Biotium, Custom CF Dye, lot 23C0811, 1:250, 4 μg/mL).

Techniques: Construct, Infection