Fold changes of candidate biomarkers were further validated by ELISA and western blotting techniques. A total of 68 serum samples were screened, including 38 OHSS women and 30 controls. ELISA quantifications for haptoglobin, fibrinogen and lipoprotein lipase (Abcam, Cambridge, MA, USA) were performed according to the manufacturers' instructions of commercial ELISA kits. For western blotting, 30 µg serum proteins were electrophoretically separated on a 10–12% SDS-PAGE gel and then transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking with 5% skimmed milk, membranes were incubated with the primary antibodies at the indicated dilutions: anti-haptoglobin (1:10,000; cat no. ab13429; Abcam), anti-fibrinogen (1:10,000; cat no. ab119948; Abcam) and anti-lipoprotein lipase (1:1,000; cat no. ab21356; Abcam) at 4°C overnight. The blots were then washed with TBS containing 0.05% Tween-20 and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000; cat no. 31439; Thermo Fisher Scientific, Inc.) at room temperature for 2 h. Immunopositive bands were visualized using Luminata Western HRP Substrates (EMD Millipore) and densitometry of the bands were estimated by FluorChem M system (ProteinSimple, San Jose, CA, USA).
Anti fibrinogen
Manufactured by Abcam
Sourced in United Kingdom
Anti-fibrinogen is a laboratory reagent used to detect and quantify the presence of fibrinogen in biological samples. Fibrinogen is a blood plasma protein that plays a crucial role in the blood clotting process. This product provides a means to analyze and measure the levels of fibrinogen, which can be important in various clinical and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Luminata western hrp substrate, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Anti ki67, by Nichirei Biosciences (1 mentions)
Anti upa, by Abcam (1 mentions)
Anti phospho c jun ser73, by Cell Signaling Technology (1 mentions)
Fv1000, by Olympus (1 mentions)
Alexa fluor 647, by Abcam (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Cytometric bead array flex set kit, by BD (1 mentions)
Lps escherichia coli 0111 b4, by Merck Group (1 mentions)
4 protocols using anti fibrinogen
1
Validating Biomarkers in OHSS Using ELISA and Western Blot
2
Histological and Immunohistochemical Analysis of Livers
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Livers were perfused with phosphate‐buffered saline and fixed in 4% paraformaldehyde overnight. Paraffin sections (4 μm) were deparaffinized and stained with hematoxylin and eosin (HE). Immunohistochemical analyses were performed by the Envision (peroxidase; Dako, Carpinteria, CA) method using anti‐Ki‐67 (Nichirei, Tokyo, Japan), anti–phospho‐c‐Jun (Ser73; Cell Signaling Technology, Danvers, MA), antifibrinogen (Abcam, Cambridge, UK), anti–α‐smooth muscle actin (α‐SMA; Dako), anti–cleaved caspase 3 (Cell Signaling Technology), anti–green fluorescent protein (GFP; ThermoFisher Scientific, Waltham, MA), antitransgelin (GeneTex, Irvine, CA), anti‐uPA (Abcam), and anti–plasminogen activator tissue‐type (tPA; Abcam) antibodies according to an antigen retrieval procedure using Target Retrieval Solution (Dako). To evaluate the extent of fibrosis, the sections were stained with sirius red F3B (Waldeck, Munster, Germany).
3
Protein Adsorption on Biomaterial Scaffolds
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Scaffolds were incubated in human blood plasma and protein solutions as described in the section “Quantitative estimation of protein adsorption“. After the washing step, proteins adsorbed to the surface of the samples were fixed with 3.7% paraformaldehyde (Sigma-Aldrich Co.) for 10 minutes and visualized by immunofluorescence (IF) technique using human-specific anti-albumin, anti-fibrinogen, and anti-fibronectin primary antibodies (Abcam, Cambridge, England). Scaffolds were incubated overnight at 4°C with primary antibodies prepared at a concentration in the range 1–5 µg/mL, washed five times with PBS, and subsequently incubated for 1 hour at room temperature with secondary antibodies conjugated to AlexaFluor®405, AlexaFluor®488, or AlexaFluor®647 (Abcam), prepared at a concentration of 2 µg/mL. Untreated scaffolds incubated with primary and secondary antibodies served as test control to check unspecific binding of antibodies to the scaffolds. Proteins adsorbed to the scaffolds were observed using a confocal laser scanning microscope (CLSM, Olympus Fluoview equipped with FV1000, Olympus, Corporation, Tokyo, Japan).
4
Antibody-mediated Fibrin Immunofluorescence
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Antibodies for injection into mice, namely, anti-mouse H-2Kd and H-2Dd IgG2a MHC-I molecules (cat. no. BE0180; clone 34-1-2S) and mouse isotype control IgG2a (cat. no. BE0085; clone C1.18.4), were obtained from Bio X Cell. For fibrin immunofluorescence staining, rabbit polyclonal anti-fibrinogen was purchased from Abcam (cat. no. ab34269). A CD41a monoclonal antibody for immunofluorescence detection of CD41 was purchased from Invitrogen (cat. no. 2072475; Thermo Fisher Scientific, Inc.). LPS (Escherichia coli 0111: B4) was obtained from Sigma-Aldrich (Merck KGaA). Bovine serum (cat. no. 22012-8612) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. Both rhodamine-conjugated goat anti-mouse antibody (cat. no. ZF-0316) and rhodamine-conjugated goat anti-rabbit antibody (cat. no. ZF-0313) were provided with Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Cytometric bead array (CBA) Flex Set kit and BCA assay kits were provided by BD Biosciences and Thermo Fisher Scientific, Inc., respectively. Mouse ELISA kits for myeloperoxidase (MPO; cat. no. ml002070), thrombin-antithrombin complex (TATc; cat. no. ml001941), tissue factor pathway inhibitor (TFPI; cat. no. ml001878) and plasminogen activator inhibitor-1 (PAI-1; cat. no. ml037410) were purchased from Miblo.
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