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.
Anti goat immpress kit
Manufactured by Vector Laboratories
The Anti-goat ImmPRESS kit is a laboratory reagent designed for the detection of goat-derived antigens or antibodies in immunohistochemical and other immunoassay applications. It contains a ready-to-use secondary antibody conjugate.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti c3d antibody, by R&D Systems (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)
Anti f4 80, by Abcam (1 mentions)
Nanozoomer s60 digital slide scanner, by Hamamatsu Photonics (1 mentions)
Anti α sma, by Abcam (1 mentions)
Tunel kit, by Roche (1 mentions)
3 protocols using anti goat immpress kit
1
Quantifying Complement Activation Markers
2
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.
3
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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