c5b 9  (Hycult Biotech)

Journal: bioRxiv

Article Title: Early Binding of Anti-Amyloid Antibodies to CAA Drives Complement Activation, Inflammation and ARIA in Mice

doi: 10.64898/2026.03.04.709591

Figure Lengend Snippet: a, Schematic of anti-Aβ immunization with 500 µg/wk 3D6 IgG2a mAb and an IgG2a isotype control for 13 weeks from 17 to 20 months in aged male APP/PS1dE9;hAPOE4 AD-like mouse model. For baseline data, baseline control mice were used. For spontaneous hemorrhage detection, 1x PBS-injected aged controls were used. b, T2*-weighted FLASH MRI of 20-month-old male APP/PS1dE9;hApoE4 mice after 13 weeks of passive immunization with 3D6 mAb (500 µg/week), showing cerebral microbleeds as hypointense spots (arrowheads), compared to 1× PBS–treated aged controls. c, Frequency of cerebral microbleeds detected by T2* MRI in 3D6–treated (n = 3) and PBS-treated (n = 4) mice. d, Quantification of Prussian blue–positive (% hemosiderin-stained) area in IgG2a and 3D6–treated mice. Unpaired t-test: **p < 0.005, ***p < 0.0005. Data represent mean ± SEM. e, Representative Prussian blue–stained sections showing hemosiderin deposits (arrowheads, with selected examples in insets) in cortex, hippocampus, thalamus, and cerebellum from 1× PBS (n=4), IgG2a isotype control (n = 5), and 3D6 IgG2a–treated mice (n = 5). Scale bar: 200 µm. f, Immunofluorescence staining in the cerebellum showing 3D6 mAb localization (anti-Ms IgG2a) and C1q deposition at sites of vascular and parenchymal amyloid labeled with Amylo-Glo (AG). g, Detection of activated complement fragments C3b/iC3b/C3c at vascular amyloid sites associated with 3D6 mAb. h, Localization of membrane attack complex (MAC/C5b-9) along vascular amyloid (arrows) and plaques (asterisk) in 3D6–treated mice. Minimal complement staining is observed in IgG2a-treated controls. Scale bars: f–h, 25 µm.

Article Snippet: Following washes in PBS, sections were blocked either in 10% normal goat serum (Gibco, #16210064), or heat inactivated goat serum (Novus Biologicals #S13110H), or normal donkey serum (Sigma, #S30-M) for 1 hour at room temperature followed by overnight 4°C incubation with primary antibodies against C1q (1:500, Abcam #ab182451), C3b/iC3b/C3c (1:300, HycultBiotech #HM1078), C5b-9 (1:300, Bioss #bs-2673R), CD31 (1:500, R&D Systems #AF3628), C3 (1:200, HycultBiotech #HM1045), GFAP (1:1000, Abcam # ab5804), MMR/CD206 (1:300,R&D #AF2535) MMP-9 (1:500, Bioss #bs-0397R), Iba-1 (1:1000; Dako, Wako #019-19741), Iba-1 (1:500, Synaptic Systems, #234308), CD68 (1:500, Bio-Rad #MCA1957), S97 Aβ (pan-Aβ rabbit polyclonal, 1:2000, gift Dr. Dominic Walsh, BWH, USA), CD31 (1:500, R&D #AF3628), Collagen-IV (1:300, Abcam #6586), Alexa Fluor 488 anti-β-Amyloid, aggregated antibody (1: 200, clone A17171C, Biolegend # 856508), Von Willebrand Factor Antibody (1:500, Novus Biologicals #NB600-586).

Techniques: Control, Injection, Staining, Immunofluorescence, Labeling, Membrane

Journal: bioRxiv

Article Title: Early Binding of Anti-Amyloid Antibodies to CAA Drives Complement Activation, Inflammation and ARIA in Mice

doi: 10.64898/2026.03.04.709591

Figure Lengend Snippet: a, Representative immunofluorescence staining in the cerebellum area showing activated C3 fragments (C3b/iC3b/C3c) and TCC/MAC complex (C5b-9) along with fibrillar amyloid (Amylo-Glo dye, AG) in IgG2a isotype and 3D6 mAb passively immunized mice. Arrows indicate iC3b or C5b-9 immunoreactivity along CAA + vessels stained with AG. Scale bar 50 µm. Quantification of % area immunoreactivity for b, iC3b c, C5b-9 per brain section. Approximately 22 to 25 Amylo-Glo + CAA vessels were outlined to quantify d, CAA + iC3b + colocalization & e, CAA + C5b9 + colocalization area. f, Serum TCC/MAC levels measured by ELISA g, % CAA estimated by AG + CD31 + colocalization data. h, Representative double IHC staining for iC3b (DAB), S97 Aβ (Vector red), with Prussian blue combination for microhemorrhages detection in the 3D6 treated mice showed iC3b-associated microhemorrhages in leptomeningeal or penetrating vessels (arrows) and signs of vascular amyloid clearance (arrowheads) in vessels with complement deposition. In IgG2a-treated mice, vascular amyloid/plaque staining is prominent with minimal complement and Prussian blue staining (arrows), Scale bar=20 µm. N=5/group, Parametric unpaired t-test, *p<0.05 and **p<0.005, ****p<0.00005. Data are expressed as mean ± SEM.

Article Snippet: Following washes in PBS, sections were blocked either in 10% normal goat serum (Gibco, #16210064), or heat inactivated goat serum (Novus Biologicals #S13110H), or normal donkey serum (Sigma, #S30-M) for 1 hour at room temperature followed by overnight 4°C incubation with primary antibodies against C1q (1:500, Abcam #ab182451), C3b/iC3b/C3c (1:300, HycultBiotech #HM1078), C5b-9 (1:300, Bioss #bs-2673R), CD31 (1:500, R&D Systems #AF3628), C3 (1:200, HycultBiotech #HM1045), GFAP (1:1000, Abcam # ab5804), MMR/CD206 (1:300,R&D #AF2535) MMP-9 (1:500, Bioss #bs-0397R), Iba-1 (1:1000; Dako, Wako #019-19741), Iba-1 (1:500, Synaptic Systems, #234308), CD68 (1:500, Bio-Rad #MCA1957), S97 Aβ (pan-Aβ rabbit polyclonal, 1:2000, gift Dr. Dominic Walsh, BWH, USA), CD31 (1:500, R&D #AF3628), Collagen-IV (1:300, Abcam #6586), Alexa Fluor 488 anti-β-Amyloid, aggregated antibody (1: 200, clone A17171C, Biolegend # 856508), Von Willebrand Factor Antibody (1:500, Novus Biologicals #NB600-586).

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Plasmid Preparation

Journal: Arthritis Research & Therapy

Article Title: Mesenchymal stem cell exosomes alleviate osteoarthritis by inhibiting complement activation via a CD59-dependent pathway

doi: 10.1186/s13075-026-03730-z

Figure Lengend Snippet: MSC exosomes attenuate complement activation and inflammation in TMJ-OA. Immunohistochemical staining for IL-1β + , TNF-⍺ + and C5b-9 + cells in ( A ) synovium and ( B ) condylar cartilage. Representative images ( n = 8 TMJs/group). Scale bars: 100–500 μm. Quantification of IL-1β + , TNF-⍺ + and C5b-9 + cells in ( C ) synovium and ( D ) condylar cartilage. The number of cells that stained positive for IL-1β, TNF-⍺ and C5b-9 were counted at 200 × magnification and expressed as the percentages of positively stained cells or positively stained cells per area. E ELISA quantification of C5b-9 in TMJ synovium and condylar cartilage. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. n = 6–8 TMJs/group

Article Snippet: P2 chondrocytes were fixed with 4% paraformaldehyde (Sigma) for 30 min, permeabilized with 0.3% Triton X-100 in PBS for 15 min, and blocked with PBS buffer containing 10% FBS and 0.1% Triton X-100 for 2 h. The cells were then incubated with primary antibodies for C5b-9 (HM3033, 1:100; Hycult Biotech) overnight at 4 °C and MMP13 (ab39012, 1:200; Abcam) for 1 h at room temperature.

Techniques: Activation Assay, Immunohistochemical staining, Staining, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Mesenchymal stem cell exosomes alleviate osteoarthritis by inhibiting complement activation via a CD59-dependent pathway

doi: 10.1186/s13075-026-03730-z

Figure Lengend Snippet: MSC exosomes inhibit complement serum-induced C5b-9 formation and MMP13 expression in a dose-dependent manner. A Immunofluorescence staining analysis of C5b-9 (green) and MMP13 (red) on chondrocytes following CS treatment with varying concentrations (1, 5, and 10 µg/ml) of MSC exosomes. Representative images ( n = 4/group). Scale bar: 100 μm. B Quantitative MFI analysis of C5b-9 and MMP13. C ELISA quantification of MMP13 in culture supernatants and ( D ) gene expression analysis of IL-6 and IL-8 following CS treatment with varying concentrations of exosomes. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, n = 4/group

Article Snippet: P2 chondrocytes were fixed with 4% paraformaldehyde (Sigma) for 30 min, permeabilized with 0.3% Triton X-100 in PBS for 15 min, and blocked with PBS buffer containing 10% FBS and 0.1% Triton X-100 for 2 h. The cells were then incubated with primary antibodies for C5b-9 (HM3033, 1:100; Hycult Biotech) overnight at 4 °C and MMP13 (ab39012, 1:200; Abcam) for 1 h at room temperature.

Techniques: Expressing, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: Arthritis Research & Therapy

Article Title: Mesenchymal stem cell exosomes alleviate osteoarthritis by inhibiting complement activation via a CD59-dependent pathway

doi: 10.1186/s13075-026-03730-z

Figure Lengend Snippet: Exosomal CD59 mediates the inhibitory effects of MSC exosomes in complement serum-induced C5b-9 formation and MMP13 expression. A Immunofluorescence staining analysis of C5b-9 (green) and MMP13 (red) on chondrocytes following CS treatment with 10 µg/ml exosomes and in the presence or absence of 10 µg/ml anti-CD59 antibody. Representative images ( n = 3/group). Scale bar: 100 μm. B Quantitative MFI analyses of C5b-9 and MMP13. C ELISA quantification of MMP13 in culture supernatants and ( D ) gene expression analysis of IL-6 and IL-8 following CS treatment with MSC exosomes and anti-CD59 antibody. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, n = 4/group

Article Snippet: P2 chondrocytes were fixed with 4% paraformaldehyde (Sigma) for 30 min, permeabilized with 0.3% Triton X-100 in PBS for 15 min, and blocked with PBS buffer containing 10% FBS and 0.1% Triton X-100 for 2 h. The cells were then incubated with primary antibodies for C5b-9 (HM3033, 1:100; Hycult Biotech) overnight at 4 °C and MMP13 (ab39012, 1:200; Abcam) for 1 h at room temperature.

Techniques: Expressing, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Gene Expression