Complement activation was determined by analysis of the complement activation products C3d, C5a, and MAC. To determine C3d deposition, immunohistochemistry staining was performed on paraffin-embedded tissue sections as previously described (Moseley et al., 2010 (link)). In brief, proteinase K enzyme antigen retrieval (Dako) was performed for 5 min at room temperature followed by peroxidase and serum blocking steps. Goat anti-C3d antibody (R&D Systems) was diluted 1:40 in PBS and incubated on slides for 2 h at room temperature, followed by antibody detection with anti–goat ImmPRESS kit (Vector Laboratories). MAC deposition was visualized by IHC on paraffin-embedded tissue using rabbit anti–rat C5b-9 antibody (1:1,000, courtesy of P. Morgan), followed by anti–rabbit ImmPRESS kit (Vector Laboratories). For C5a determinations, liver homogenates were prepared from frozen liver samples homogenized in cell lysis buffer (Sigma-Aldrich) containing a protease inhibitor cocktail (Thermo Fisher Scientific). Homogenates were centrifuged at 10,000 g for 10 min at 4°C, and C5a levels in supernatant determined using an ELISA kit according to the manufacturer’s instructions (R&D Systems, BD). Infiltrating neutrophils were quantified by MPO content of liver samples using a MPO ELISA kit (Hycult Biotechnology) according to the manufacturer’s instructions.
Goat anti c3d antibody
Manufactured by R&D Systems
Sourced in United States
The Goat anti-C3d antibody is a laboratory reagent used for the detection and analysis of the C3d complement protein. It is a polyclonal antibody raised in goats against the C3d fragment of the C3 complement component. The antibody can be used in various immunological techniques, such as Western blotting, ELISA, and immunohistochemistry, to identify and quantify the presence of C3d in biological samples.
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Lab products found in correlation
Anti goat immpress kit, by Vector Laboratories (3 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Cell lysis buffer, by Merck Group (1 mentions)
Immpress anti rabbit kit, by Vector Laboratories (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
T per buffer, by Thermo Fisher Scientific (1 mentions)
Anti f4 80, by Abcam (1 mentions)
Nanozoomer s60 digital slide scanner, by Hamamatsu Photonics (1 mentions)
Anti α sma, by Abcam (1 mentions)
Odyssey system, by LI COR (1 mentions)
Mouse anti gapdh, by Cell Signaling Technology (1 mentions)
Rabbit anti claudin 1, by Abcam (1 mentions)
Mouse anti nf κb p65 antibody, by Abcam (1 mentions)
Rabbit anti tlr4 antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti iba1 antibody, by Fujifilm (1 mentions)
Mouse anti gfap, by Cell Signaling Technology (1 mentions)
Tunel kit, by Roche (1 mentions)
5 protocols using goat anti c3d antibody
1
Quantifying Complement Activation Markers
2
Quantitative Western Blot Analysis of Retinal Proteins
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Protein extraction from the retinal tissue was carried out using T-PER buffer (Thermo Fisher Scientific) with the addition of 1% protease and 1% phosphatase inhibitor (Sigma-Aldrich). Protein samples were separated by SDS-PAGE using denatured, reducing conditions. The proteins were transferred onto a PVDF membrane. The membrane was blocked with blocking buffer (5% bovine serum albumin (BSA) and 0.1% Tween 20 in Tris-buffered saline) and primary antibodies (goat anti-C3d antibody (Cat. #AF2655; R&D Systems) or rabbit anti-GAPDH antibody (Cat. #CSB-PA00025A0Rb; Cusabio Technology LLC, Houston, TX, USA)) were added overnight. Detection was performed using secondary antibodies (anti-goat IgG-POD #SBA-6442-05, anti-rabbit-POD #711-546-152 (Dianova GmbH, Hamburg, Germany)) and the Lumi-Light Western Blotting Substrate (Cat. # 12015200001; Sigma-Aldrich) and imaged by the FluorChem FC2 Imaging System (AppliChem, Darmstadt, Germany). Western blot images were edited in Adobe Photoshop CS6 (San Jose, CA, USA) and analysed for signal intensity using ImageJ software (National Institutes of Health).
3
Histological Evaluation of Liver Tissue
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Fresh liver tissues were frozen, and sliced into 8 μm thick sections. The sections were stained with an Oil Red O staining. Liver biopsy specimens were also fixed with 10% neutral formalin and embedded in paraffin, and then tissue sections (5 μm thick) were stained with hematoxylin–eosin (H&E) and Sirius Red. Histopathological changes in the liver biopsies were assessed using a NanoZoomer S60 digital slide scanner (Hamamatsu, Japan).
Paraffin-embedded sections (4 μm thick) were processed for immune-histochemical staining. Antigen retrieval was performed by pressure cooking for 5 min in citrate buffer (pH 6), followed by peroxidase and serum blocking steps. The sections were incubated with goat anti-C3d antibody (R&D Systems, 1:100 dilution), anti-F4/80 (CST, 1:500 dilution) or anti-α-SMA (Abcam, 1:500 dilution) for 2 h at room temperature, followed by antibody detection with an anti-goat ImmPRESS kit (Vector Laboratories). The images were collected using the NanoZoomer S60 digital slide scanner.
Paraffin-embedded sections (4 μm thick) were processed for immune-histochemical staining. Antigen retrieval was performed by pressure cooking for 5 min in citrate buffer (pH 6), followed by peroxidase and serum blocking steps. The sections were incubated with goat anti-C3d antibody (R&D Systems, 1:100 dilution), anti-F4/80 (CST, 1:500 dilution) or anti-α-SMA (Abcam, 1:500 dilution) for 2 h at room temperature, followed by antibody detection with an anti-goat ImmPRESS kit (Vector Laboratories). The images were collected using the NanoZoomer S60 digital slide scanner.
4
Protein Expression Analysis of Ischemic Penumbra
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Fifty milligrams of ischemic penumbra tissues (n = 6) was used for Western blot analysis as previously described [15 (link)]. The primary antibodies include the following: mouse anti-VE-cadherin (1:1000, Cell Signaling Technology), rabbit anti-AQP4 (1:1000, Proteintech, USA), rabbit anti-Claudin-1 (1:1000, Abcam, USA), mouse anti-GFAP (1:1000, Cell Signaling Technology), rabbit anti-Iba1 antibody (1:500, Wako), goat anti-C3d antibody (1:1000, RD Systems), rabbit anti-S100A10 (1:1000; Invitrogen), rabbit anti-TLR4 antibody (1:1000, Cell Signaling Technology), mouse anti-phospho-NF-κB p65 (Ser536) antibody (1:1000, Abcam), mouse anti-NF-κB p65 antibody (1:1000, Abcam), mouse anti-GAPDH (1:1000, Cell Signaling Technology). The membranes were then incubated with the corresponding HRP-conjugated secondary antibody for 2 h. Protein bands were visualized using the LI-COR Odyssey System (LI-COR Biotechnology, USA).
5
Immunohistochemical Analysis of Liver Tissues
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The resected liver tissues were fixed overnight with 10% formalin, and paraffin-embedded sections (4-μm thick) were prepared. Sections were then processed for immunohistochemical staining. Antigen retrieval was performed by pressure cooking for 3 min in citrate buffer (pH 6), followed by peroxidase and serum blocking steps. The sections were incubated with goat anti-C3d antibody (R&D Systems, 1:100 dilution) for 2 h at room temperature, followed by antibody detection with an anti-goat ImmPRESS kit (Vector Laboratories). Apoptosis was quantified by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) using a TUNEL kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. The cell nuclei were counterstained with DAPI (1 mg/mL). The apoptotic cells were labeled green with the TUNEL staining kit. The images were collected using a fluorescent microscope (IX-71; Olympus, Tokyo, Japan).
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