mouse anti c3  (Proteintech)

Journal: Journal of Extracellular Vesicles

Article Title: Yersinia enterocolitica O:3 Outer Membrane Vesicles as a Platform for Complement Activation

doi: 10.1002/jev2.70270

Figure Lengend Snippet: (A) The impact of OMVs on germicidal activity of serum against YeO3‐c bacteria. The influence of OMVs‐associated with: LPS polysaccharide chain length (a) and pYV‐coded factors (b). Data from one of at least two experiments with the use of separately prepared culture supernatants with similar results are presented. 1 sterile cell culture supernatants (secreted by bacteria grown to OD 600 = 0.6) were used as a source of OMVs; 2 expression at 37°C only; 3 NHS inactivated 30 min at 56°C. (B) Y. enterocolitica O:3 OMV‐induced complement activation in the presence of calcium and magnesium chelators. ELISA plates were coated with 10 8 of OMVs secreted by YeS‐c bacteria grown at 4°C, 22°C and 37°C and with OMVs of YeRa‐c, YeRd1‐c and YeRe‐c variants propagated at 37°C. After incubation with/without EDTA, EGTA or EGTA/Mg 2+ (EGTA supplemented with Mg 2+ ions), products of C3 activation were detected with specific antibodies. Data from one of two experiments with similar results are presented. (C) Comparison of complement activation and MBL binding by Yersinia enterocolitica O:3 bacterial cells, LPS and OMVs. ELISA plates were coated with 50 ng/well of bacteria, LPS or OMVs. The deposition of C3 (a, possible AP, CP and LP involvement), C4 (b, possible CP and LP involvement) or C4 LP‐dependent (d) activation products, TCC formation (c, possible AP, CP and LP involvement) and MBL‐binding (e) was analyzed after preincubation with normal human serum (filled columns) or with EDTA‐treated NHS (stripped columns) or without serum (open columns, negative control). Data from one of two experiments with similar results are presented. Dots represent individual OD values for each well. (D) Recognition of Y. enterocolitica O:3 OMVs by human mannose‐binding lectin. YeS‐c_37°C bacteria (1), OMVs (2) and LPS (3) were separated in SDS‐PAGE. After transfer to nitrocellulose and incubation with NHS, the bound MBL was detected with specific mAb (a). The experiment was performed at least 5 times. To control the loading of bacteria, LPS or OMVs the presence of OPS in separated samples was confirmed with anti‐6‐deoxy‐L‐altropyranose mAbs (b).

Article Snippet: Depletion of functional C3 from serum was verified in Western blot [acc. to Younger et al. ( )], using goat antibodies against mouse C3 (MP Biomedicals, USA), HRP‐conjugated anti‐goat Ig (Dako) for detection of intact C3 α‐chain and ECL detection system.

Techniques: Activity Assay, Bacteria, Sterility, Cell Culture, Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Comparison, Binding Assay, Negative Control, SDS Page, 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 acute single-dose immunization in 19-month-old APP NL-G-F KI mice (n=3-4/group) with biotinylated 3D6 or IgG2a mAb (25 mg/kg, i.p.), followed by euthanasia at 24 h. b, Brain sections stained with Amylo-Glo (AG) for amyloid, Collagen-IV for blood vessels’ basement membrane, Streptavidin 555 for biotinylated 3D6 or IgG2a, and endothelial marker CD31 in IgG2a and 3D6-biotinylated mAb-injected mice; images taken from the leptomeningeal penetrating vessels along the cortical pial surface. Arrowheads point towards CAA-bound Streptavidin 555, indicative of biotinylated mAb binding; asterisks indicate plaques. c–d , Brain sections labeled with AG and Streptavidin-AF555 showing biotinylated mAb distribution in c, leptomeningeal vessels, and d, large parenchymal arterioles. Arrowheads indicate CAA-associated mAb binding and C1q deposition observed in 3D6-treated mice. e, Schematic of acute immunization in 25-month-old APP NL-G-F KI mice (n= 7-9/group) treated with anti-Aβ 3D6 IgG2a mAb or IgG2a isotype control. f, Brain sections stained for aggregated Aβ, anti-mouse IgG2a (to detect 3D6 and IgG2a), and C1q in IgG2a control- and 3D6-treated mice. Arrows indicate cerebellar meningeal vessel-associated labeling; asterisks mark parenchymal plaque labeling. Adjacent sections were stained with Prussian blue (counterstained with nuclear fast red) for microhemorrhages and H&E for RBC extravasation. g, Triple labeling with AG, anti-mouse IgG2a, and collagen-IV to distinguish plaque (AG⁺AntiMsIgG2a⁺ColIV⁻) and vascular (AG⁺AntiMsIgG2a⁺ColIV⁺) mAb binding in 3D6-treated brain sections. h, Quantification of % cerebellar plaque vs CAA labeling of mAb in a subset of 3D6 treated mice (n=5), *p < 0.05, paired t-test. Scale bar: b=50 µm; c,d,f =100 µm; g = 250 µm.

Article Snippet: After three PBS-Tween washes, biotinylated goat anti-mouse C3 antibody (1:1000, MP Biomedicals #55463 biotinylated as described above for anti-C1q) was added and incubated for 1 hour at 37°C.

Techniques: Staining, Membrane, Marker, Injection, Binding Assay, Labeling, Control