Journal: Annals of Translational Medicine

Article Title: Characteristics of pre-sensitization-related acute antibody-mediated rejection in a rat model of orthotopic liver transplantation

doi: 10.21037/atm-22-4311

Figure Lengend Snippet: Levels of C4d were increased in sensitized followed transplant rats. (A) Lymphocytes of LEW rats were isolated for C4d detect using anti-C4d antibody and secondary antibody with FITC. (B) Flow data are presented on the statistical graph (n=3). Significant differences between each group were marked by ***, P<0.001, ****, P<0.0001. LEW, Lewis; FITC, fluorescein isothiocyante.

Article Snippet: At 14 days after liver transplantation, splenic cells of LEW rats (recipients) were isolated and detected for C4d expression using rabbit anti-rat C4d (Cat. No. HP8034-100UG; Hycult Biotech, Beutelsbach, Germany) and goat anti-rabbit IgG Antibody (FITC conjugate) (Cat. No. AP132F; Millipore, Darmstadt, Germany).

Techniques: Isolation

Journal: Annals of Translational Medicine

Article Title: Characteristics of pre-sensitization-related acute antibody-mediated rejection in a rat model of orthotopic liver transplantation

doi: 10.21037/atm-22-4311

Figure Lengend Snippet: Accelerate liver rejection and C4d deposition occurred in sensitized followed transplant rats. (A) Inflammation of portal areas, bile ducts, vascular endothelium, and capillaries is shown by HE staining in each group. (B) C4d deposition is shown in IHC. LEW, Lewis; HE, hematoxylin and eosin; IHC, immunohistochemistry.

Article Snippet: At 14 days after liver transplantation, splenic cells of LEW rats (recipients) were isolated and detected for C4d expression using rabbit anti-rat C4d (Cat. No. HP8034-100UG; Hycult Biotech, Beutelsbach, Germany) and goat anti-rabbit IgG Antibody (FITC conjugate) (Cat. No. AP132F; Millipore, Darmstadt, Germany).

Techniques: Staining, Immunohistochemistry

Journal: Annals of Translational Medicine

Article Title: Characteristics of pre-sensitization-related acute antibody-mediated rejection in a rat model of orthotopic liver transplantation

doi: 10.21037/atm-22-4311

Figure Lengend Snippet: AMR was occurred in sensitized followed transplant rats

Article Snippet: At 14 days after liver transplantation, splenic cells of LEW rats (recipients) were isolated and detected for C4d expression using rabbit anti-rat C4d (Cat. No. HP8034-100UG; Hycult Biotech, Beutelsbach, Germany) and goat anti-rabbit IgG Antibody (FITC conjugate) (Cat. No. AP132F; Millipore, Darmstadt, Germany).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Binding of Complement Inhibitor C4b-binding Protein to a Highly Virulent Streptococcus pyogenes M1 Strain Is Mediated by Protein H and Enhances Adhesion to and Invasion of Endothelial Cells

doi: 10.1074/jbc.m113.502955

Figure Lengend Snippet: FIGURE 1. Protein H binds C4BP thus degrading C4b and reducing C3b deposition on S. pyogenes as well as increasing adherence to and invasion of endothelial cells. For analysis of complement activation and opsonization S. pyogenes strains AP1, MC25, BM27.6, and BMJ71 were incubated in increasing concentrations of NHS for 1 h at 37 °C. Subsequently, bacteria were stained with the respective antibodies and subjected to flow cytometry analysis to asses C4BP binding (A) and C3b deposition on the bacterial surface (B). C4BP bound to protein H serves as factor I cofactor in degradation of C4b (C). Immobilized protein H was incubated with 20 g C4BP, washed thoroughly, and then incubated with Factor I and 125I-C4b as indicated. As control, soluble purified proteins were used. Subsequently, samples were separated by SDS-PAGE. Because BSA cannot bind C4BP, even in the presence of factor I, C4b degradation could not be observed (C, top). S. pyogenes AP1 and BM27.6 were incubated with 100 g of C4BP, washed thoroughly, and then incubated with factor I and 125I-C4b as indicated. Only AP1 binding C4BP was able to support degradation of C4b (C, lower panel) as indicated by the appearance of the C4d band. BM27.6 unable to bind C4BP could nearly not degrade C4b. S. pyogenes AP1 and BM27.6 were preincubated with 10% NHS and subsequently stained with antibodies for C4d and C4d. Flow cytometry analysis revealed that AP1 had decreased levels C4c compared with C4d, indicating that C4c was released by cleavage. BM27.6 had comparable values of C4c and C4d judged by mean fluorescence intensities (D) confirming that cleavage did not occur. S. pyogenes AP1 and BM27.6 were preincubated with 25 g of C4BP for 30 min and surplus C4BP was removed. Bacteria were then incubated with HUVECs at an multiplicity of infection of 25 at 37 °C for 3 h to let S. pyogenes adhere. After removing all unbound S. pyogenes, remaining bacteria were plated on blood agar plates and counted (E). To assess invasion,priortoplating,extracellularbacteriawerekilledusinggentamicinandpenicillin(F).S. pyogenesstrainsfeaturingproteinHshowstrongC4BPbinding but decreased deposition of C3b as well as increased adherence to and invasion of endothelial cells. Results from at least three independent experiments are shown. In C and D, representative experiments are shown. Error bars indicate S.D. values. Statistical significance is shown: *, p 0.05 or ****, p 0.0001 by two-way analysis of variance. ns, not significant; neg con, negative control; pos con, positive control.

Article Snippet: The following antibodies were used for detection, blocking, and binding assays: -C4BPMK104, MK102, MK96 andMK67 (27), -C3d (A0063, Dako), -C4c (A211, Quidel), -C4d (A213, Quidel), and mouse isotype IgG1 and IgG2a (ImmunoTools).

Techniques: Activation Assay, Incubation, Bacteria, Staining, Flow Cytometry, Binding Assay, Control, Purification, SDS Page, Fluorescence, Infection, Negative Control, Positive Control

Journal: Journal of Clinical Investigation

Article Title: Complement activation on endothelium initiates antibody-mediated acute lung injury

doi: 10.1172/jci138136

Figure Lengend Snippet: Figure 7. High endothelial HLA class I expression in human lungs and complement activation after transfusion in clinical TRALI. Samples of human lungs were dissociated for measurement of cell surface HLA class I (HLA-ABC) expression using flow cytometry. (A) Gating strategy, (B) representative histograms of fully stained cells relative to FMO controls, and (C) quantification of HLA class I expression on different cells expressed as MFI (D–G) (n = 4). EC, endothelial cell. Plasma content of the classical/lectin pathway complement component C4 and its activation and degradation product C4d were mea- sured using pre- and posttransfusion samples from patients diagnosed with TRALI or control patients who did not develop TRALI after transfusion. (D) Concentrations of C4d and (E) fold change in C4d after transfusion, and (F) C4d/C4 ratios and (G) fold change in C4d/C4 ratios after transfusion. Normal- ized data (E and G) provided for enhanced display of individual changes after transfusion. Mean (D and F, bars) or median (E and G, lines) and individual values (D and F). In D and F, a 2-way repeated-measures mixed-model approach was used with pretransfusion values set as covariates, followed by Holm’s multiplicity correction for comparisons of effect of TRALI development after transfusion and effect of transfusion within the control group and the TRALI group. Control group, n = 19; TRALI group, n = 9. P values < 0.05 are shown.

Article Snippet: ELISAs for mouse C3 (ab157711, Abcam), human C4 (ab108824, Abcam), and human C4d (A009, Quidel) were used according to manufacturers’ instructions.

Techniques: Expressing, Activation Assay, Flow Cytometry, Staining, Clinical Proteomics, Control

Journal: Journal of Clinical Investigation

Article Title: Complement activation on endothelium initiates antibody-mediated acute lung injury

doi: 10.1172/jci138136

Figure Lengend Snippet: Figure 8. Anti-HLA antibodies in lung transplant recipients activate complement on pulmonary endothelium and increase risk of CLAD or death. Serial sections of a transbronchial biopsy from a lung transplant recipient diagnosed with antibody-mediated rejection (AMR) showing (A) diffuse, strong linear microvascular endothelial C4d positivity (immunohistochemistry: C4d = brown, hematoxylin counterstain = blue), and (B) organizing acute lung injury with increased neutrophils (H&E staining). (C) Chest computed tomography (CT) scan from lung transplant recipient diagnosed with AMR showing bilateral consolidations. (D and E) Kaplan-Meier curves showing chronic lung allograft dysfunction–free (CLAD-free) survival over time in lung transplant recipients comparing effects of (D) number of episodes of anti-HLA donor-specific antibodies (DSAs) (adjusted curves from Cox proportional hazards model using serologic detection of DSAs as a time-dependent variable), or (E) different reactivities of de novo anti-HLA DSAs (actual CLAD-free survival within groups). (D and E) P values are from log-rank tests from whole Cox models, n = 215 with 15 patients with anti-HLA class I DSAs only, 22 with anti-HLA class II DSAs only, and 7 with detected DSAs against both HLA classes.

Article Snippet: ELISAs for mouse C3 (ab157711, Abcam), human C4 (ab108824, Abcam), and human C4d (A009, Quidel) were used according to manufacturers’ instructions.

Techniques: Immunohistochemistry, Staining, Computed Tomography

Journal: Journal of Clinical Immunology

Article Title: Testing the Activity of Complement Convertases in Serum/Plasma for Diagnosis of C4NeF-Mediated C3 Glomerulonephritis

doi: 10.1007/s10875-016-0290-5

Figure Lengend Snippet: Mechanism of convertase stabilization by C4NeF isolated from patient seven. a Spontaneous decay of classical C3 convertase assembled from purified components. Sensitized erythrocytes were coated with C1 and C4. Addition of C2 initiated the process of convertase formation. Convertase activity was assessed at indicated time points in the same manner, as described in Figs. and . b The same experiment as in a but with CD55-Fc added together with C2. c Representative blot of the C4b degradation pattern (C4b, factor I, and C4BP present) and comparison to negative control with no factor I added and sample, in which C4b cleavage was inhibited by excessive anti-C4c antibody. d Quantification of C4d produced upon C4b degradation in the presence of Igs isolated from patient seven or from NHS. When anti-C4c antibodies were tested in the same experiments, C4d readout was below the lower level of detection and therefore is not shown. All graphs show data collected from at least 3 independent experiments and statistical significance was assessed with two-way ANOVA assay at * p < 0.05, ** p < 0.0,1 and *** p < 0.001

Article Snippet: Human sera depleted from C3 and/or C5 as well as mouse anti- C4d antibody (A253) were purchased from Quidel.

Techniques: Isolation, Purification, Activity Assay, Comparison, Negative Control, Produced

Journal: Clinical and Experimental Immunology

Article Title: Complement activation in leprosy: a retrospective study shows elevated circulating terminal complement complex in reactional leprosy

doi: 10.1111/cei.12767

Figure Lengend Snippet: Antibodies for the Meso Scale discovery (MSD) assays.

Article Snippet: The multiplex set comprised C1s, the activation markers C4d, Bb, iC3b and TCC and the regulator Factor H (FH) (using FH Y402, FH H402 monoclonal antibodies which quantifies the concentration of total FH, as described previously 26 ) and clusterin (Table ). table ft1 table-wrap mode="anchored" t5 Table 2 caption a7 Assay Coating antibody (source) Detection antibody (source) C1s M81 (Hycult) F33 (in‐house, B. P. M.) C4d Neo C4d (A251, Quidel) C4d (A213, Quidel) Bb Neo Bb (A252, Quidel) JC1 (in‐house, B. P. M.) iC3b Neo iC3b (Hycult) C3‐30 (in‐house, B. P. M.) TCC aE11 (Hycult) E2 (in‐house, B. P. M.) FH Y402 MBI‐6 (in‐house, B. P. M.) Ox‐24 FH H402 MBI‐7 (in‐house, B. P. M.) Ox‐24 Clusterin Polyclonal anti‐apolipoprotein J (AB825, Millipore) MBI‐40 (in‐house, B. P. M.) Open in a separate window TCC = terminal complement complex; FH = Factor H. Antibodies for the Meso Scale discovery (MSD) assays.

Techniques:

Journal: bioRxiv

Article Title: Single-cell atlas of pig-to-monkey kidney xenotransplantation reveals macrophage chimerism and an IFN-ε orchestrated graft protective immune niche

doi: 10.64898/2026.03.21.713416

Figure Lengend Snippet: (A) Haematoxylin and eosin-based evaluation for xenotransplantation control #1 (sections a1-a3) and #2 (sections c1-c3), xenograft #1 (sections b1-b3) and #2 (d1-d3). Fields representative of glomeruli (row 1), peritubular capillaries (row 2), and tubulointerstitial compartments (row 3) are shown. Black arrows show non-infiltrated peritubular capillaries (sections a2 and c2), and infiltrated peritubular capillaries (section b2 and d2). (B) Immunohistochemistry-based evaluation for xenotransplantation control #1 (sections a1-a3) and #2 (sections c1-c3), xenograft #1 (sections b1-b3) and #2 (d1-d3). Fields representative of IgM (row 1), IgG (row 2) and C4d (row 3) stainings are shown. (C) Immunofluorescence staining of infiltrated macrophages with signature proteins. Scale bars, 100 µm.

Article Snippet: The primary antibody panel including: anti-IgM (11016-1-AP, 1:100, Proteintech), anti-IgG (ab109489, 1:500, Abcam), C4d (22233-1-AP, 1:200, Proteintech), anti-CD31 (ab28364, 1:50, Abcam), anti-vWF (ab6994, 1:200, Abcam), anti-CD3 (GB12014, 1:1000, Servicebio) and anti-CD15 (YT0726, 1:200, Immunoway) for IHC; anti-CD68 (bs-0649R, 1:200, Bioss), anti-SPP1 (83341-1-RR, 1:200, Proteintech), anti-VEGFA (66828-1-Ig, 1:200, Proteintech), anti-PD-L1 (ab205921, 1:200, Abcam), anti-IDO1 (ab211017, 1:1000, Abcam), anti-ARG1 (66129-1-Ig, 1:200, Proteintech), anti-FCGR2B (NB100-65338, 1:100, Novus Biologicals), anti-CD8a (66868-1-Ig, 1:400, Proteintech), and anti-KLRD1 (84466-5-RR, 1:200, Proteintech) for multiplexed IF.

Techniques: Control, Immunohistochemistry, Immunofluorescence, Staining

Journal: Frontiers in medicine

Article Title: Overactivation of the complement system may be involved in intrarenal arteriolar lesions in IgA nephropathy.

doi: 10.3389/fmed.2022.945913

Figure Lengend Snippet: FIGURE 1 Three pathways of complement activation. The classical pathway is activated by IgG– and/or IgM–containing immune complexes. The lectin pathway requires a particular sugar moiety pattern to be recognized and bound by MBL/Ficolins/Collectin 11, leading to a classical pathway–like activation cascade. C4d represents the activation of classical pathway or/and lectin pathway. The alternative pathway is constantly initiated by spontaneous hydrolysis of C3 [C3b (H2O)] that is efficiently powered by the covalent attachment of C3b on an activating surface. Complement factor H (FH) and factor H related protein 5 (FHR5) are regulator proteins of alternative pathway. Three pathways lead to formation of C3 convertase and converge at the C3 level. The addition of C3b to the C3 convertase creates C5 convertase, which triggers the formation of the terminal pathway complete complex (C5b-9), which is also known as membrane attack complex.

Article Snippet: Detail staining protocols were optimized for frozen renal biopsy tissue and formalin-fixed paraffin-embedded renal biopsy tissue with the following antibodies: mouse monoclonal anti-human FH antibody (Santa Cruz, United States), and rabbit polyclonal antihuman FHR-5 antibody (GeneTex, California, United States), mouse monoclonal anti-human MBL antibody (clone 3E7) (Hycult Biotech, United States), rabbit polyclonal anti-human C4d antibody (Bio-Rad, United States), mouse monoclonal anti-human C5b-9 antibody (Abcam, United States), rabbit polyclonal anti-human C3c (Dako, Glostrup, Denmark), rat monoclonal anti-human Gd-IgA1 antibody (KM55) (Immuno-Biological Laboratories, Japan) (11, 15).

Techniques: Activation Assay, Membrane

Journal: Frontiers in medicine

Article Title: Overactivation of the complement system may be involved in intrarenal arteriolar lesions in IgA nephropathy.

doi: 10.3389/fmed.2022.945913

Figure Lengend Snippet: FIGURE 7 Positive staining for MBL (A,G), C4d (B,H), FH (C,I), FHR5 (D,J), C3c (E,K), and MAC (F,L) by immunohistochemistry and immunofluorescence, respectively, along intrarenal arterioles in the arteriolar lesions group of IgAN. (Original magnification ×200).

Article Snippet: Detail staining protocols were optimized for frozen renal biopsy tissue and formalin-fixed paraffin-embedded renal biopsy tissue with the following antibodies: mouse monoclonal anti-human FH antibody (Santa Cruz, United States), and rabbit polyclonal antihuman FHR-5 antibody (GeneTex, California, United States), mouse monoclonal anti-human MBL antibody (clone 3E7) (Hycult Biotech, United States), rabbit polyclonal anti-human C4d antibody (Bio-Rad, United States), mouse monoclonal anti-human C5b-9 antibody (Abcam, United States), rabbit polyclonal anti-human C3c (Dako, Glostrup, Denmark), rat monoclonal anti-human Gd-IgA1 antibody (KM55) (Immuno-Biological Laboratories, Japan) (11, 15).

Techniques: Staining, Immunohistochemistry