Journal: Journal of thrombosis and haemostasis : JTH

Article Title: The lectin-like domain of thrombomodulin interferes with complement activation and protects against arthritis.

doi: 10.1111/j.1538-7836.2006.02033.x

Figure Lengend Snippet: Fig. 4. Complement C3 deposition in joints. Healthy 5–8-week old female TMwt/wt (A) and TMLeD/LeD (B) mice were killed and knee joints were processed for immunohistochemical staining to detect complement factor C3 deposition. These sections are representative of three mice. No C3 was detectable in the joints of TMwt/wt mice (A). C3 was readily observed on the articular surface (small arrows) and in the bone marrow space of TMLeD/LeD mice (large arrows) (B). Higher power views of the articular surface are shown in the right upper corner of each panel.

Article Snippet: Specific immunostaining of histologic sections was achieved by overnight incubation with the following primary antibodies: CD45 (rat, 1:100; BecktonDickinson, Erembodegem, Belgium, no. 553076);Mac3 (rat, 1:300; BecktonDickinson, no. 553322); MPO (rabbit, 1:100, Dako A/S, Glostrup, Denmark, no. A0398); HMGB1 (rabbit; 1:250, Santa Cruz Biotechnology, Boechout, Belgium, no. sc12523); and complement factor C3 (rat, 1:200; Cedarlane,Uden, theNetherlands, no. CL7503AP), which recognizes intact C3, C3b, iC3b and C3d, but not C3a.

Techniques: Immunohistochemical staining, Staining

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Endothelial Cell–Specific Inactivation of TSPAN12 (Tetraspanin 12) Reveals Pathological Consequences of Barrier Defects in an Otherwise Intact Vasculature

doi: 10.1161/ATVBAHA.118.311689

Figure Lengend Snippet: Complement deposition and histopathologic changes in retinas of aged Tspan12 ECKO mice. A , Complement factor C4 deposition around retinal blood vessels, inner nuclear layer (INL), and outer plexiform layer (OPL). White arrows point to some of the small cystoid lesions decorated with complement. B , H&E staining of a control retina and a relatively strongly affected area of a mutant retina reveals cystoid lesions consistent with edema. C , Increased F4/80 staining on microglia of mutant retinas. D through F , Extravastion of IgG, transferrin, and albumin in Tspan12 ECKO retinas. G , COL4 (collagen IV) staining in retinal blood vessels of 12-mo-old ECKO mice is increased, staining in the inner limiting membrane (white arrowheads) serves as internal staining control.

Article Snippet: The following primary antibodies and lectins were used for this study: GS Isolectin B4 Alexa-488 conjugate 1:100 (Invitrogen, I21411), rat PECAM (platelet endothelial cell adhesion molecule) 1:50 (BD, 550274), rat PLVAP 1:50 (BD, 550563), rabbit desmin 1:200 (Cell Signaling, D93F5), rat vascular endothelial (VE)-cadherin 1:100 (BD, clone 11D4), rat complement component C4 1:200 (Cedarlane, CL7504AP), goat transferrin 1:200 (R&D, AF3987), rabbit COL4 (collagen IV) 1:200 (Abcam, 6586), goat albumin 1:200 (Bethyl, A90-234A), goat IBA1 1:200 (Novus, NB100-1028), rat F4/80 1:200 (AbD Serotec, MCA497R), rabbit PKC (protein kinase C) α 1:200 (Santa Cruz, C-20), and rabbit Sox9 1:200 (Millipore, AB5535).

Techniques: Staining, Control, Mutagenesis, Membrane

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Endothelial Cell–Specific Inactivation of TSPAN12 (Tetraspanin 12) Reveals Pathological Consequences of Barrier Defects in an Otherwise Intact Vasculature

doi: 10.1161/ATVBAHA.118.311689

Figure Lengend Snippet: Colocalization of complement factor C4 with several retinal cell types. A , Twelve-mo-old Tspan12 ECKO retinal cross-sections stained with anti-C4 and IB4. Split and merged channels are shown. Arrows indicate blood vessels decorated with C4. B , Colocalization of extravasated IgG and C4 on a subset of retinal cells and on the borders of cystoid lesions in the mutant tissue. C , Rod-bipolar cell spacing seems increased in the mutant tissue. The section shows also 1 or 2 C4-decorated Müller glia cells, which were recognized by their extensions towards the outer limiting membrane (arrow). D , Disorganized Müller cells in the mutant tissue. Sox9 marks nuclei of Müller glia. Arrow highlights a C4-decorated cell which was recognized as amacrine cell based on cell body location and cell shape. E , Partial colocalization of C4 with microglia marker IBA-1. PKC indicates protein kinase C; and Sox9, SRY-box 9.

Article Snippet: The following primary antibodies and lectins were used for this study: GS Isolectin B4 Alexa-488 conjugate 1:100 (Invitrogen, I21411), rat PECAM (platelet endothelial cell adhesion molecule) 1:50 (BD, 550274), rat PLVAP 1:50 (BD, 550563), rabbit desmin 1:200 (Cell Signaling, D93F5), rat vascular endothelial (VE)-cadherin 1:100 (BD, clone 11D4), rat complement component C4 1:200 (Cedarlane, CL7504AP), goat transferrin 1:200 (R&D, AF3987), rabbit COL4 (collagen IV) 1:200 (Abcam, 6586), goat albumin 1:200 (Bethyl, A90-234A), goat IBA1 1:200 (Novus, NB100-1028), rat F4/80 1:200 (AbD Serotec, MCA497R), rabbit PKC (protein kinase C) α 1:200 (Santa Cruz, C-20), and rabbit Sox9 1:200 (Millipore, AB5535).

Techniques: Staining, Mutagenesis, Membrane, Marker

Journal: Nature Communications

Article Title: A comprehensive synthetic library of poly- N -acetyl glucosamines enabled vaccine against lethal challenges of Staphylococcus aureus

doi: 10.1038/s41467-024-47457-4

Figure Lengend Snippet: A Complement deposition tests were performed as described using pooled sera from rabbits ( n = 2 per group) immunized with mQβ–PNAG conjugates, the 5GlcNH 2 –TT conjugate, or from a sample of pooled normal human sera. Titers and 95% confidence intervals (CI) were determined by linear regression using log 10 values of the average of replicate serum dilutions to determine the X intercept and 95% CI when Y = 0.5 (OD 405 nm of ELISA plate reading). P values indicate the significance of the deviation of the slope of the titration curve from zero to identify sera with activity at P < 0.05. mQβ–PNAG10 and mQβ–PNAG26 conjugates were more potent than the mQβ–PNAG0 and 5GlcNH 2 –TT conjugate in inducing C1q deposition onto purified PNAG. Normal human serum had no significant C1q depositing activity in spite of having a binding titer to PNAG (see Fig. ) consistent with prior reports that naturally acquired human antibody to PNAG is not functional due to the inability to activate the complement pathway , . Titers were determined by simple linear regression. B Pooled sera from rabbits ( n = 2 per group) immunized with mQβ–PNAG conjugate led to significantly higher levels of opsonic killing activities against S. aureus cells. Three aliquots were prepared from each pooled serum and the individual values of the three aliquots were presented. Source data are provided as a file.

Article Snippet: Subsequently, affinity‐purified goat anti‐human complement C1q antibody (Cedarlane, Inc, CL7341AP, 100 μL, 1:1000 dilution in GVB) was added to each well and incubated for 1 h under ambient temperature.

Techniques: Enzyme-linked Immunosorbent Assay, Titration, Activity Assay, Purification, Binding Assay, Functional Assay