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 7-week treatment in 16.5-month-old APP/PS1dE9;hAPOE4 mice (n = 7-8/group) with 3D6 or IgG2a control mAb. b, Prussian blue-labeled hemosiderin deposits in cortex indicating microhemorrhage (asterisks). Scale bar: 50 µm. c, Quantification of Prussian blue–positive area (%) in the whole sagittal brain section; Mann-Whitney test, p < 0.0005. d, Amylo-Glo (AG)–positive fibrillar amyloid area (%) in cortex; unpaired t-test, p < 0.0005. e, Prussian blue staining with eosin counterstain in cortex showing hemosiderin deposits (asterisks) and red-orange cytoplasm of extravasated RBCs (arrows). Scale bar: 100 µm. f, Immunostaining with anti-mouse IgG2a fluorescently-tagged secondary antibody, C1q and AG in brain sections from 3D6- and IgG2a-treated mice. Scale bar: 50 µm. Representative immunofluorescence staining in the g, cortex and h, cerebellum showing C1q and activated C3 fragments C3b/iC3b/C3c deposition near AG-positive plaques (asterisks) and vascular amyloid (arrow), Scale bar in g & h : 250 µm. Quantification of % immunoreactivity (IR) for i, C1q; j, C1q + CD31 + AG + colocalized area; k, activated C3 fragments C3b/iC3b/C3c; and l, activated C3 fragments + CD31 + AG + colocalization in cerebellum. m, Levels of complement C3 in the plasma following 7-weeks of passive immunization. Unpaired t-test in j, k and Mann-Whitney test for i, l . *p<0.05 and **p<0.005, ***p<0.0005. 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: Control, Labeling, MANN-WHITNEY, Staining, Immunostaining, Immunofluorescence, Clinical Proteomics

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: Blood Vessels, Thrombosis & Hemostasis

Article Title: A second generation of C3 humanized rats for preclinical evaluation of human C3 inhibitors

doi: 10.1016/j.bvth.2026.100138

Figure Lengend Snippet: The second-generation hC3 rats exhibit normal renal function but show signs of glomeruli iC3b deposition. Urine samples were collected from WT and second-generation hC3 rats at 2 and 6 months of age. Urea nitrogen and creatinine levels were measured using a Roche Cobas 6000 analyzer (A-D). Male and female second-generation hC3 rats at 8 months of age were perfused and kidneys were collected to detect iC3b by confocal imaging using an iC3b neoepitope-specific monoclonal antibody, showing some positive signals in the glomeruli (E-H). MDA-MB-231 cells incubated with 10% normal human serum were processed in the identical way to serve as a positive control (I-J). 2nd gen, second generation; Ms IgG, mouse IgG, which was used as the staining negative control.

Article Snippet: Sections were incubated overnight at 4°C with mouse anti-human iC3b antibody (1:100 dilution; Neoantigen/Bio-Rad, catalog no. MCA2607) prepared in 1% BSA.

Techniques: Imaging, Incubation, Positive Control, Staining, Negative Control

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 7-week treatment in 16.5-month-old APP/PS1dE9;hAPOE4 mice (n = 7-8/group) with 3D6 or IgG2a control mAb. b, Prussian blue-labeled hemosiderin deposits in cortex indicating microhemorrhage (asterisks). Scale bar: 50 µm. c, Quantification of Prussian blue–positive area (%) in the whole sagittal brain section; Mann-Whitney test, p < 0.0005. d, Amylo-Glo (AG)–positive fibrillar amyloid area (%) in cortex; unpaired t-test, p < 0.0005. e, Prussian blue staining with eosin counterstain in cortex showing hemosiderin deposits (asterisks) and red-orange cytoplasm of extravasated RBCs (arrows). Scale bar: 100 µm. f, Immunostaining with anti-mouse IgG2a fluorescently-tagged secondary antibody, C1q and AG in brain sections from 3D6- and IgG2a-treated mice. Scale bar: 50 µm. Representative immunofluorescence staining in the g, cortex and h, cerebellum showing C1q and activated C3 fragments C3b/iC3b/C3c deposition near AG-positive plaques (asterisks) and vascular amyloid (arrow), Scale bar in g & h : 250 µm. Quantification of % immunoreactivity (IR) for i, C1q; j, C1q + CD31 + AG + colocalized area; k, activated C3 fragments C3b/iC3b/C3c; and l, activated C3 fragments + CD31 + AG + colocalization in cerebellum. m, Levels of complement C3 in the plasma following 7-weeks of passive immunization. Unpaired t-test in j, k and Mann-Whitney test for i, l . *p<0.05 and **p<0.005, ***p<0.0005. Data are expressed as mean ± SEM.

Article Snippet: Sections were then incubated overnight at 4°C with primary antibody against C3b/iC3b/C3c (1:300, HycultBiotech #HM1078).

Techniques: Control, Labeling, MANN-WHITNEY, Staining, Immunostaining, Immunofluorescence, Clinical Proteomics

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: Sections were then incubated overnight at 4°C with primary antibody against C3b/iC3b/C3c (1:300, HycultBiotech #HM1078).

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: Sections were then incubated overnight at 4°C with primary antibody against C3b/iC3b/C3c (1:300, HycultBiotech #HM1078).

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

Journal: Scientific Reports

Article Title: CFD protein deficiency induce slow transit constipation is correlated with gut microbial dysbiosis

doi: 10.1038/s41598-026-41597-x

Figure Lengend Snippet: The colon of Cfd –/– mice had inflammatory cell infiltration and high inflammatory factor expression. ( A ) Histopathological structure of small intestine, and colon of WT and Cfd –/– mice. ( B ) IL-17 and IL-6 mRNA levels in the colon were measured by real-time PCR, n = 6. ( C ) Representative IHC staining of MUC2 in colon of WT and Cfd –/– mice. ( D ) Representative images of immunofluorescence analysis and quantitation of C3 cleavage product (C3b, iC3b, C3c) deposition in colon of WT and Cfd –/– mice. * P < 0.05; ** P < 0.01.

Article Snippet: Then, incubated with rabbit anti-C3b, iC3b, C3c (1:300; Hycult, HM1065) at 4°C overnight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Immunofluorescence, Quantitation Assay