anti cd54 antibody  (Bio-Rad)

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: HeLa cells stably expressing EndoA3-GFP (green) and transiently expressing ICAM1-mScarlet (red) were imaged for 2 min using live-cell TIRF microscopy, with 1-s intervals between frames. Representative of two independent experiments. Scale bar: 10 µm.

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: LB33-MEL cells stably expressing EndoA3-GFP (green) and transiently expressing ICAM1-mScarlet (red) were imaged for 2 min using live-cell TIRF microscopy, with 1-s intervals between frames. Representative of two independent experiments. Scale bars: 10 μm (full-size image) and 2 μm (enlarged cropped area).

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: HeLa cells stably expressing EndoA3-GFP (green) and transiently expressing ICAM1-mScarlet (red) were imaged for 2 min using live-cell TIRF microscopy, with 1-s intervals between frames. The punctate ICAM1 structure is indicated by a white arrowhead. Representative of two independent experiments. Scale bar: 10 µm.

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: LB33-MEL cells stably expressing EndoA3-GFP (green) and transiently expressing ICAM1-mScarlet (red) were imaged for 2 min using live-cell TIRF microscopy, with 1-s intervals between frames. The punctate ICAM1 structure is indicated by a white arrowhead. Representative of two independent experiments. Scale bars: 10 μm (full-size image) and 2 μm (enlarged cropped area).

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: HeLa cells stably expressing EndoA3-GFP (green) and transiently expressing ICAM1-mScarlet (red) were imaged for 2 min using live-cell TIRF microscopy, with 1-s intervals between frames. Representative of two independent experiments. Scale bars: 10 μm (full-size image) and 2 μm (enlarged cropped area).

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: LB33-MEL cells stably expressing EndoA3-GFP (green) and transiently expressing ICAM1-mScarlet (red) were imaged for 2 min using live-cell TIRF microscopy, with 1-s intervals between frames. Representative of two independent experiments. Scale bar: 10 µm.

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: LB33-MEL cells stably expressing EndoA3-GFP (green) and transiently expressing ICAM1-mScarlet (red) were imaged for 2 min using live-cell TIRF microscopy, with 1-s intervals between frames. The punctate ICAM1 structure is indicated by a white arrowhead. Representative of two independent experiments. Scale bar: 10 µm.

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: A HeLa cell transiently expressing ICAM1-EGFP (green) forming an immune synapse-like conjugate with a CD8 T cell (red) was imaged by live-cell spinning-disk confocal microscopy, for 3 min, with 1-s intervals between frames. ICAM1-positive tubulo-vesicular carriers are observed fusing at the ICAM1-enriched contact zone, which grows and expands over time. Notably, anterograde transport of ICAM1-positive carriers toward the contact zone appears stronger than retrograde transport, and carrier density is higher in the region of the HeLa cell engaged in the immune synapse compared with regions not involved in synapse formation. Associated tracking and quantification data are shown in Figure 4C–G . Representative of two independent experiments. Scale bar: 20 µm.

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: A HeLa cell transiently expressing ICAM1-EGFP (green) forming an immune synapse-like conjugate with a CD8 T cell (red) was imaged by live-cell spinning-disk confocal microscopy, for 3 min, with 1-s intervals between frames. ICAM1-positive tubulo-vesicular carriers are observed fusing at the ICAM1-enriched contact zone (white arrows), which grows and expands over time. Representative of two independent experiments. Scale bar: 5 µm.

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: ( A ) Illustration of the SNAP-tag-based BG-labeled antibody uptake assay to study membrane protein endocytosis and retrograde transport. ( B ) Confocal images of GalT-GFP-SNAP (green) and TMR-Star (red) in HeLa cells stably expressing the Golgi-resident GFP-fused SNAP-tag construct (HeLa GalT-GFP-SNAP). Actin (phalloidin, white) and nuclei (DAPI, blue) were also stained. Fluorescence intensity profile was made along the dashed line region in enlarged cropped area and shows the colocalization of both signals. Scale bar: 20 μm. ( C–F ) Retrograde transport of ALCAM and ICAM1. Continuous BG-labeled anti-ALCAM ( C, E ) and anti-ICAM1 ( D, F ) antibody uptake for 4 h at 37°C in HeLa GalT-GFP-SNAP cells. ( C, D ) Western blot analysis of HeLa GalT-GFP-SNAP cells transfected for 72 h with siRNAs: negative control (siCtrl) or against retromer subunits (siVPS35 and siVPS26A). Immunodetection made with anti-SNAP, anti-VPS35, anti-VPS26A, and anti-α-Tubulin (loading control) antibodies. Quantification of the covalent IgG-SNAP-GFP-GalT complex is shown as fractions of siCtrl condition (histogram). Quantification of VPS35 and VPS26A depletion is shown in . ( E, F ) Western blot analysis of HeLa GalT-GFP-SNAP cells transfected for 72 h with siRNAs: negative control (siCtrl) or against EndoA3 (siEndoA3). Immunodetection made with anti-SNAP, anti-EndoA3, and anti-clathrin heavy chain (CHC, loading control) antibodies. Quantification of the covalent IgG-SNAP-GFP-GalT complex (IB:anti-SNAP) is shown as fractions of siCtrl condition (histogram). Quantification of EndoA3 depletion is shown in . Data information: In ( B ), images are from a single experiment. Quantification data ( C–F ) are pooled from three independent experiments. Data are presented as mean ± SEM. *p<0.05, **p<0.01. One-sample t test and Wilcoxon test. Figure 1—source data 1. Original files for western blot analyses displayed in . Figure 1—source data 2. PDF files containing original western blots for .

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques: Labeling, Membrane, Stable Transfection, Expressing, Construct, Staining, Fluorescence, Western Blot, Transfection, Negative Control, Immunodetection, Control

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: ( A ) Confocal images of GalT-GFP-SNAP (green) and TMR-Star (red) in LB33-MEL cells stably expressing the Golgi-resident GFP-fused SNAP-tag construct (LB33-MEL GalT-GFP-SNAP). Actin (phalloidin, white) and nuclei (DAPI, blue) were also stained. The fluorescence intensity profile was made along the dashed line region in the enlarged cropped area and shows the colocalization of both signals. Scale bar: 20 μm. ( B ) Confocal images of GalT-GFP-SNAP (green) and TGN46 (red) in LB33-MEL GalT-GFP-SNAP cells. Nuclei (DAPI, blue) were also stained. The fluorescence intensity profile was made along the dashed line region in enlarged cropped area and shows the colocalization of both signals, indicating that GalT-GFP-SNAP is correctly localized at the TGN . Scale bar: 10 μm. ( C ) Quantifications of the immunoblots shown in (IB: anti-VPS35 and anti-VPS26A) confirm depletion efficiency of VPS35 and VPS26A in HeLa GalT-GFP-SNAP cells. ( D ) Retrograde transport of ICAM-1. Continuous BG-labeled anti-ICAM1 antibody uptake for 4 h at 37°C in LB33-MEL GalT-GFP-SNAP cells. Western blot analysis of LB33-MEL GalT-GFP-SNAP cells transfected with siRNAs: negative control (siCtrl) or against retromer subunits (siVPS35 and siVPS26A). Immunodetection with anti-SNAP, anti-VPS35, anti-VPS26A, and anti-α-Tubulin (loading control) antibodies. Quantification of the covalent IgG-SNAP complex is shown as fractions of siCtrl condition (histogram). Quantification of VPS35 and VPS26A depletion is shown in ( E ). ( E ) Quantifications of the immunoblots shown in ( D ) confirm depletion efficiency of VPS35 and VPS26A in LB33-MEL GalT-GFP-SNAP cells. ( F ) Retrograde transport of ALCAM. Continuous BG-labeled anti-ALCAM antibody uptake for 4 h at 37°C in HeLa GalT-GFP-SNAP cells. Western blot analysis of HeLa GalT-GFP-SNAP cells transfected for 72 h with siRNAs: negative control (siCtrl) or against Rab6 (siRab6). Immunodetection made with anti-SNAP, anti-Rab6, and anti-clathrin heavy chain (CHC, loading control) antibodies. Quantification of the covalent IgG-SNAP-GFP-GalT complex is shown as fractions of siCtrl condition (histogram). Quantification of Rab6 depletion is shown in ( G ). ( G ) Quantifications of the immunoblots shown in ( F ) confirm depletion efficiency of Rab6 in HeLa GalT-GFP-SNAP cells. ( H ) Quantifications of the immunoblots shown in (IB: anti-EndoA3) confirm depletion efficiency of EndoA3 in HeLa GalT-GFP-SNAP cells. ( I ) Retrograde transport of ALCAM. Continuous BG-labeled anti-ALCAM antibody uptake for 4 h at 37°C in LB33-MEL GalT-GFP-SNAP cells. Western blot analysis of LB33-MEL GalT-GFP-SNAP cells transfected (or not) with plasmids encoding free GFP (GFP+) or EndoA3-GFP (EndoA3+). Immunodetection with anti-SNAP, anti-EndoA3, and anti-α-Tubulin (loading control) antibodies. Quantification of the covalent IgG-SNAP-GFP-GalT complex is shown as fractions of the GFP +condition (histogram). Data information: In ( A, B ), images are from a single experiment. In ( C ), data are pooled from six independent experiments. In ( D–H ), data are pooled from three independent experiments. In ( I ), data are pooled from two independent experiments. Data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. One-sample t test and Wilcoxon test. Figure 1—figure supplement 1—source data 1. Original files for western blot analyses displayed in . Figure 1—figure supplement 1—source data 2. PDF files containing original western blots for .

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques: Stable Transfection, Expressing, Construct, Staining, Fluorescence, Western Blot, Labeling, Transfection, Negative Control, Immunodetection, Control

Journal: bioRxiv

Article Title: IL-1β and TNF drive endothelial dysfunction and coagulopathy in acute COVID-19

doi: 10.64898/2026.03.21.713333

Figure Lengend Snippet: (A-D) NHBE/HMVEC-L co-cultures were infected with SARS-CoV-2 (MOI 1) or mock-infected, and cells were fixed at 24, 48, and 72h post-infection. A) Immunofluorescence staining of ICAM-1 (green), F-actin (phalloidin; grey), and nuclei (DAPI; blue) in HMVEC-L in the basal compartment of the co-culture. Images are representative of three independent experiments. B) Quantification of HMVEC-L ICAM-1 intensity analysed by 2-way ANOVA with Sidak’s multiple comparison’s test. C) Immunofluorescence staining of VE-cadherin (magenta) in HMVEC-L in the basal compartment of the co-culture. Images are representative of three independent experiments. D) Quantification of the percentage of gaps in the endothelial monolayer was analysed by 2-way ANOVA with Sidak’s multiple comparison’s test. E-H) NHBE/HMVEC-L co-cultures were infected with SARS-CoV-2 (MOI 1) or mock-infected, and cells were fixed at 72h post-infection. E) Immunofluorescence staining of Zombie Red (magenta) in HMVEC-L in the basal compartment of the co-culture. Images are representative of four independent experiments. F) The percentage of Zombie Red-positive cells between conditions was analysed using the Kruskal-Wallis test with Dunn’s multiple testing correction. G) Immunofluorescence staining and imaging of CellTrace Yellow-labelled platelets (yellow), VE-cadherin (magenta), and nuclei (DAPI; blue) in HMVEC-L in the basal compartment of the co-culture. Images are representative of three independent experiments. H) Gaps in the cellular monolayer were outlined relative to VE-cadherin staining, and only the platelets present in gaps were quantified. I) The number of platelets (CellTrace Yellow positive particles larger than 2 mm) in gaps was quantified and analysed by the Kruskal-Wallis test with Dunn’s multiple testing correction. Scale bar for all images = 50 µm. 5 ROIs per experiment were quantified (small dots) and are colour-coded per experiment. The average of the 5 ROIs is represented by the large dot (colour-coded by experiment), and the data show the mean ± SEM. Asterisks indicate statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were then incubated overnight at 4 °C with primary antibodies against mouse ICAM-1 (BioXCell; BE0020-1) and mouse CD31 (New England Biolabs; 77699S).

Techniques: Infection, Immunofluorescence, Staining, Co-Culture Assay, Imaging

Journal: bioRxiv

Article Title: IL-1β and TNF drive endothelial dysfunction and coagulopathy in acute COVID-19

doi: 10.64898/2026.03.21.713333

Figure Lengend Snippet: (A-E) NHBE/HMVEC-L co-cultures infected with SARS-CoV-2 (MOI 1) and then treated with 100 mg/mL dexamethasone or media alone immediately post-infection. Cells were fixed at 72h post-infection. A) Immunofluorescence staining of ICAM-1 (green) in HMVEC-L in the basal compartment of the co-culture. Images are representative of 2 independent experiments. B) Quantification of HMVEC-L ICAM-1 intensity, where data shows mean + SD. C) Immunofluorescence staining of VE-cadherin (magenta) in HMVEC-L in the basal compartment of the co-culture. Images are representative of three independent experiments. D) Quantification of the percentage of gaps in the endothelial monolayer was analysed by one-way ANOVA with Sidak’s multiple comparison’s test. E) Viral titres from the apical compartment of SARS-CoV-2-infected NHBE/HMVEC-L co-cultures, untreated or treated with 100 mg /mL dexamethasone, at 72h post-infection. n = 3 independent experiments, analysed by unpaired, two-way t -test. F) Schematic of supernatant transfer experiment. G-J) NHBE monocultures were infected with SARS-CoV-2 for 48h. The supernatant from the basal compartment was then transferred onto HMVEC-L. 100 mg /mL dexamethasone or PBS was added to the NHBE basal supernatants before they were transferred onto HMVEC-L. After 24h, HMVEC-L were fixed for immunofluorescence staining. G) Immunofluorescence staining of ICAM-1 (green) in HMVEC-L. Images are representative of 3 independent experiments. H) Quantification of HMVEC-L ICAM-1 intensity analysed by one-way ANOVA with Sidak’s multiple comparison’s test. I) Immunofluorescence staining of VE-cadherin (magenta) in HMVEC-L. Images are representative of three independent experiments. J) Quantification of the percentage of gaps in the endothelial monolayer was analysed by one-way ANOVA with Sidak’s multiple comparison’s test. Scale bar for all images = 50 µm. 5 ROIs per experiment were quantified (small dots) and are colour-coded per experiment. The average of the 5 ROIs is represented by the large dot (colour-coded by experiment), and the data show the mean ± SEM. Asterisks indicate statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were then incubated overnight at 4 °C with primary antibodies against mouse ICAM-1 (BioXCell; BE0020-1) and mouse CD31 (New England Biolabs; 77699S).

Techniques: Infection, Immunofluorescence, Staining, Co-Culture Assay

Journal: bioRxiv

Article Title: IL-1β and TNF drive endothelial dysfunction and coagulopathy in acute COVID-19

doi: 10.64898/2026.03.21.713333

Figure Lengend Snippet: (A-H) NHBE/HMVEC-L co-cultures were infected with SARS-CoV-2 (MOI 1) or mock-infected and treated with 10 mg/mL anti-TNF (Adalimumab) immediately post-infection. Cells were fixed at 72h post-infection. A) Immunofluorescence staining for ICAM-1 (green), F-actin (Phalloidin; grey), and nuclei (DAPI; blue). Images are representative of n = 3 independent experiments. B) ICAM-1 intensity between conditions was analysed by one-way ANOVA, with Sidak’s multiple comparison test. C) Immunofluorescence staining for VE-cadherin (magenta), F-actin (Phalloidin; grey), and nuclei (DAPI; blue). Images are representative of n = 3 independent experiments. D) Quantification of gaps in the endothelial monolayer under different conditions was determined by calculating the percentage of the image area covered by gaps, and analysed by one-way ANOVA, with Sidak’s multiple comparison’s test. E) Zombie Red-stained cells (magenta) indicate cells (containing F-actin and nuclei) undergoing cell death. Images are representative of n = 3 independent experiments. F) The percentage of Zombie Red positive cells was analysed by one-way ANOVA, with Sidak’s multiple comparison’s test. G) Immunofluorescent staining of CellTrace Yellow-labelled platelets incubated with HMVEC-L. Images are representative of n = 3 independent experiments. H) The number of platelets (CellTrace Yellow positive particles larger than 2 mm) in gaps was quantified and analysed by the Kruskal-Wallis test with Dunn’s multiple testing correction. I) NHBE monocultures and NHBE/HMVEC-L co-cultures were infected with SARS-CoV-2 (MOI 1) or mock-infected, and TNF levels in the apical and basal supernatants were analysed at 24, 48, and 72h post-infection. Data show the mean ± SEM of 3 independent experiments, analysed by 2-way ANOVA. (J-M) NHBE monocultures were infected with SARS-CoV-2 for 48h. The supernatant from the basal compartment was then transferred onto HMVEC-L. Anti-TNF (10 mg/mL) or PBS was added to the NHBE basal supernatants before they were transferred onto HMVEC-L. After 24h, HMVEC-L were fixed for immunofluorescence staining. J) Immunofluorescence staining of ICAM-1 (green) in HMVEC-L. Images are representative of 3 independent experiments. K) Quantification of HMVEC-L ICAM-1 intensity was analysed by one-way ANOVA with Sidak’s multiple comparison’s test. L) Immunofluorescence staining of VE-cadherin (magenta) in HMVEC-L. Images are representative of three independent experiments. M) Quantification of the percentage of gaps in the endothelial monolayer was analysed by one-way ANOVA with Sidak’s multiple comparison’s test. Scale bar for all images = 50 µm. 5 ROIs per experiment were quantified (small dots) and are colour-coded per experiment. The average of the 5 ROIs is represented with the large dot (colour-coded per experiment), and the data shows mean ± SEM. Asterisks indicate statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were then incubated overnight at 4 °C with primary antibodies against mouse ICAM-1 (BioXCell; BE0020-1) and mouse CD31 (New England Biolabs; 77699S).

Techniques: Infection, Immunofluorescence, Staining, Comparison, Incubation

Journal: bioRxiv

Article Title: IL-1β and TNF drive endothelial dysfunction and coagulopathy in acute COVID-19

doi: 10.64898/2026.03.21.713333

Figure Lengend Snippet: (A-F) NHBE/HMVEC-L co-cultures were infected with SARS-CoV-2 (MOI 1) or mock-infected and treated with 10 mg/mL Anakinra immediately post-infection. Cells were fixed at 72h post-infection. A) Immunofluorescence staining for ICAM-1 (green), F-actin (Phalloidin; grey), and nuclei (DAPI; blue). Images are representative of n = 5 independent experiments. B) ICAM-1 intensity between conditions was analysed by one-way ANOVA, with Sidak’s multiple comparison test. C) Immunofluorescence staining for VE-cadherin (magenta), F-actin (Phalloidin; grey), and nuclei (DAPI; blue). Images are representative of n = 3 independent experiments. D) Quantification of gaps in the endothelial monolayer under different conditions was determined by calculating the percentage of the image area covered by gaps, and analysed by one-way ANOVA, with Sidak’s multiple comparison’s test. E) Zombie Red-stained cells (magenta) indicate cells (containing F-actin and nuclei) undergoing cell death. Images are representative of n = 3 independent experiments. F) The percentage of Zombie Red positive cells was analysed by one-way ANOVA, with Sidak’s multiple comparison’s test. G) IL-1β levels in the apical supernatant of NHBE monocultures infected with SARS-CoV-2 (MOI 1) or mock-infected, at 24, 48, and 72h post-infection (n = 3 independent experiments). H) TNF levels in the apical (left panel) and basal (right panel) supernatant of SARS-CoV-2-infected NHBE/HMVEC-L co-cultures, untreated or treated with Anakinra, at 24, 48, and 72h post-infection (n = 2 independent experiments). Scale bar for all images = 50 µm. 5 ROIs per experiment were quantified (small dots) and are colour-coded per experiment. The average of the 5 ROIs is represented with the large dot (colour-coded per experiment), and the data shows mean ± SEM. Asterisks indicate statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were then incubated overnight at 4 °C with primary antibodies against mouse ICAM-1 (BioXCell; BE0020-1) and mouse CD31 (New England Biolabs; 77699S).

Techniques: Infection, Immunofluorescence, Staining, Comparison

Journal: bioRxiv

Article Title: IL-1β and TNF drive endothelial dysfunction and coagulopathy in acute COVID-19

doi: 10.64898/2026.03.21.713333

Figure Lengend Snippet: A) Representative immunohistochemistry (IHC) images of lungs from SARS-CoV-2-infected (10 4 TCID50) or mock-infected wild-type (WT), Tnf -/- , Il1b -/- , and Tnf -/- IL1b -/- mice, harvested at 3 days post-infection (dpi). Tissues were stained with CD31 (magenta), ICAM-1 (green), and DAPI (blue). Scale bar = 50 µm. Images are representative of 5 images per mouse, 3 mice per group. B) Quantification of ICAM-1 intensity in areas of CD31 staining, analysed by 2-way ANOVA with Sidak’s multiple testing correction. 5 ROIs per mouse were quantified (small dots) and are colour-coded per mouse. The average of the 5 ROIs is represented with the large dot (colour-coded per mouse), and the data shows mean ± SEM. C-F) Aged K18-hACE c57BL/6□J mice infected with SARS-CoV-2 (10 3 PFU) and treated with an isotype control or anti-IL-1β antibody at 1h or 3 days post-infection. Lungs were harvested at day 6 post-infection. C) Lung tissues were stained with CD31 (magenta), ICAM-1 (green), and DAPI (blue). Scale bar = 100 µm. Images are representative of 5 images per mouse, 4-6 mice per group. D) Quantification of ICAM-1 intensity in areas of CD31 staining, analysed by one-way ANOVA, with Sidak’s multiple comparison’s test. 5 ROIs per mouse were quantified (small dots) and are colour-coded per mouse. The average of the 5 ROIs is represented by the large dot (colour-coded per mouse), and the data show the mean ± SEM. E) Lung tissues were stained for fibrinogen. Scale bar = 50 µm. Images are representative of 4-6 mice per group. F) Quantification of fibrinogen staining intensity analysed by one-way ANOVA, with Sidak’s multiple comparison’s test. Asterisks indicate statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were then incubated overnight at 4 °C with primary antibodies against mouse ICAM-1 (BioXCell; BE0020-1) and mouse CD31 (New England Biolabs; 77699S).

Techniques: Immunohistochemistry, Infection, Staining, Control