Plasma levels of c-reactive protein (CRP) were measured using commercially available ELISA kits (Sigma-Aldrich, St. Louis, Missouri, USA). The levels of 8-isoprostanes, prostaglandin E2 (PGE2), thromboxane B2 (Cayman, Ann Arbour, MI), resolvin E1 (MyBiosource, San Diego, USA), fibrinogen (Abcam, Cambridge, Massachusetts, USA), human CC10/CC16 (MyBiosource, San Diego, USA), (human synonym name for mouse CC16 is CC10), pentraxin 3 (Sigma, St. Louis, USA), Intracellular Adhesion Molecule-1 (RD Systems, Minneapolis, USA), desmosine (MyBiosource, San Diego, USA), NT-proBNP (RD Systems, Minneapolis, USA) and malondialdehyde (MDA) (Cell Biolabs, San Diego, USA) were measured quantitatively by commercially available ELISA kits as per manufacturer’s instructions
Fibrinogen
Manufactured by Abcam
Sourced in United States, United Kingdom
Fibrinogen is a soluble plasma glycoprotein that plays a crucial role in the blood clotting process. It is a key component in the formation of fibrin, which is the structural basis of blood clots. Fibrinogen is produced by the liver and is an essential part of the coagulation cascade.
Automatically generated - may contain errors
Lab products found in correlation
Desmosine, by MyBioSource (2 mentions)
Thromboxane b2, by Cayman Chemical (1 mentions)
Nt probnp, by R&D Systems (1 mentions)
Prostaglandin e2, by Cayman Chemical (1 mentions)
F4 80, by Bio-Rad (1 mentions)
Pd l1, by BioLegend (1 mentions)
Resolvin d1, by Cayman Chemical (1 mentions)
Resolvin d2, by Cayman Chemical (1 mentions)
Resolvin e1, by Cayman Chemical (1 mentions)
C reactive protein (crp), by Merck Group (1 mentions)
Icam 1, by R&D Systems (1 mentions)
Mayer s hematoxylin solution, by Fujifilm (1 mentions)
Alexa fluor 488 anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Anti rat ig hrp detection kit, by BD (1 mentions)
Bz 2 analyzer, by Keyence (1 mentions)
Biorevo bz 9000, by Keyence (1 mentions)
Laminin, by Abcam (1 mentions)
Elastase 1, by Merck Group (1 mentions)
Elastase 2, by Abcepta (1 mentions)
15 protocols using fibrinogen
1
Measuring Inflammatory and Oxidative Biomarkers
2
Imaging Tumor Microenvironment Immune Markers
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Frozen sections of the primary tumor and liver metastases were fixed with 4% paraformaldehyde in PBS and blocked with blocking solution (5% horse sera and 1% goat sera in PBS). Immunohistochemical staining of tissue sections was performed using antibodies against PD-L1 (BioLegend, San Diego, CA, USA), CD31 (BD Biosciences, San Jose, CA, USA), fibrinogen (Abcam, Cambridge, MA, USA), and various immune cells, such as CD8 (Invitrogen, Waltham, MA, USA), F4/80 (Abd Serotec, Raleigh, NC, USA), and CD45 (BD Pharmingen, San Diego, CA, USA). To image delivery of iv injected rat anti-PD-L1 IgG or isotype control IgG in tumors, the tissue sections were incubated with Alexa-fluor 647-labeled anti-rat IgG (Jackson ImmunoResearch, West Grove, PA, USA). All images were acquired using laser scan confocal microscopy (Nikon Inc., Tokyo, Japan). ImageJ software (NIH, Bethesda, MD, USA) was used to calculate the area fractions of positive staining on the captured fluorescence images [15 ]. All graphs are plotted based on the area fractions, and each symbol represents an individual imaging analysis.
3
Quantifying Amyloid, vWF, and Fibrinogen in Tumor Samples
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Levels of murine Amyloid β42, (Fisher Scientific), vWF and fibrinogen (both from Abcam) were measured in plasma collected from the skin and pancreatic tumor-bearing mice or in serum-free CMed from pancreatic CAFs as per manufacturer’s instructions. Human platelet-poor plasma was obtained by centrifuging whole blood from healthy donors at 2000 × g for 15 min. Samples were immediately snap frozen until use. Archived melanoma patient plasma samples (MELRESIST NRES: 11.NE.0312) and healthy controls were measured for levels of Amyloid β42 (Abcam) as per manufacturers guidelines.
4
Measuring inflammatory biomarkers by ELISA
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ELISAs for PGE2 (Cat #514010, Cayman Chemical Company, Ann Arbor, MI), fibrinogen (cat# ab208036, Abcam) and CRP (Cat # DCRP00, R&D Systems, Minneapolis, MN)21 (link) were performed according to the manufacturers’ instructions.
5
Measurement of Inflammatory Biomarkers
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Plasma levels of biomarkers were measured using commercially available ELISA kits: C-reactive protein (CRP) (Sigma, St Louis, MO, USA), desmosine (MyBiosource, San Diego, CA, USA), 8-isoprostane, prostaglandin E2, thromboxane B2, resolvin D1, resolvin D2, resolvin E1, leukotriene E4 (Cayman, Ann Arbor, MI, USA), fibrinogen (Abcam, Cambridge, MA, USA), intracellular adhesion molecule (ICAM)-1 (R&D Systems), 4-hydroxynonenal, malondialdehyde (Cell Biolabs, San Diego, CA, USA) and lipoxin A4 (TSZ ELISA, Waltham, MA, USA). Urine samples were analysed for 8-isoprostane, leukotriene E4, desmosine and 8-oxo-dG (HT 8-Oxo-dG Human ELISA; Trevigen, Gaithersburg, MD, USA). Salivary biomarkers IL-1β, IL-6, prostaglandin E2, resolvin D1 and resolvin D2 were also measured. EBC samples were analysed for 8-isoprostane. All these biomarkers were measured quantitatively as per the manufacturers' instructions.
6
Immunofluorescence and Immunohistochemistry of Platelets
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For immunofluorescent, the cryosection (10 μm) was fixed with 4% paraformaldehyde, and pemeabilized with 0.1% Triton X-100. After blocking with 1% bovine serum albumin, antibodies to PDPN (clone: D2-40, DAKO, Glostrup, Denmark) and Fibrinogen (1:100) (Abcam) were incubated overnight. The slides were subsequently stained with secondary antibodies; 4 μg/ml of Alexa Fluor 594 anti-mouse IgG (H + L) and Alexa Fluor 488 anti-rabbit IgG (H + L) (Thermo Fisher Scientific), respectively. The mounted slides with ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific) were examined using BioRevo BZ-9000 (Keyence, Osaka, Japan). For immunohistochemistry, the cryosection (10 μm) was fixed in ice-cold acetone and endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol. After blocking with 10% goat serum, an antibody to CD41 (1:100) (clone: MWReg30, GeneTex, Irvine, CA, USA) was incubated overnight. Colouring was performed with Anti-Rat Ig HRP Detection Kit (BD Pharmingen). Mayer’s hematoxylin solution (Wako) was used for counterstain. All images were taken with BioRevo BZ-9000 (Keyence). CD41-positive area was analysed by BZ-II Analyzer (Keyence).
7
Immunoblot and Immunoprecipitation Analysis
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Immunoblotting and immunoprecipitation were performed as previously described (65 (link)). Equal amounts of tissue lysates were analyzed, and the blots were incubated with specific antibodies to elastase 1 (Sigma-Aldrich), elastase 2 (Abgent), kallikrein 1 (Sigma-Aldrich), kallikrein 2 (Abgent), kallikrein 5 (Acris Antibodies), kallikrein 6 (Sigma-Aldrich), and collagen I, II, III, IV, fibronectin, fibrinogen and laminin (all from Abcam) as previously described (65 (link)). Beta-Actin (Sigma-Aldrich) was used as a loading control.
8
Lung Immunohistochemistry Protocol
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The mice were sacrificed under deep anesthesia and perfused transcardially with phosphate-buffered saline (PBS) and then 4% paraformaldehyde in PBS (pH 7.4). For immunohistochemistry, only the left lung, comprising a single lobe, was utilized. The isolated left lungs were soaked in 4% paraformaldehyde in PBS for 48 h and then in a cryoprotective solution (30% sucrose). The resulting fixed lung tissues were embedded in Scigen Tissue-Plus™ O.C.T. Compound (Thermo Fisher Scientific, Waltham, MA, United States) and then frozen at −80°C. Cryosections (14 μm thick) obtained from the lung were used for immunostaining for GSNOR (Thermo Fisher Scientific, cat. #: 11051-1-AP), CD31 (Thermo Fisher Scientific, cat. #. 14045285), ICAM-1 (Thermo Fisher Scientific, cat. #: MA5407), VCAM-1 (Thermo Fisher Scientific, cat. #: MA5-11447), CD11b (Thermo Fisher Scientific, cat. #: PA5-79532), Ly-6G (Cell Signaling, Danvers, MA, United States, cat. #: 31469s), fibrin (GeneTex, cat. #: GTX19079) and fibrinogen (Abcam, cat. #: ab92572). All digital images were taken using a BX-60 microscope equipped with a DP70 digital camera unit (Olympus, Tokyo, Japan).
9
Quantification of Spinal Cord Proteins in EAE Mice
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EAE mice were sacrificed on Day 17 postimmunization. The spinal cords were harvested and homogenized. After centrifugation at 12,000×g for 15 min, the supernatant was aspirated. Then, 5X loading buffer was added, and the protein was boiled in a 100°C metal bath for 10 min to denature. The protein was separated by SDS–PAGE and transferred onto a PVDF membrane (Merck KGaA, Darmstadt, Germany). After blocking, the membranes were incubated with primary antibodies against fibrinogen (1:1000, Abcam), iNOS (1:1000, Abcam), and GAPDH (1:10000, Abcam). Subsequently, the membranes were washed with TBST and incubated with an HRP‐labeled secondary antibody (1:10000, Jackson Immuno). The membranes were imaged on a Bio‐Rad (Bio‐Rad, Hercules, CA), and the image information was analyzed with ImageJ.
10
ADAMTS‐13 Variant Expression and Fibrinogen Cleavage
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ADAMTS‐13 variants were expressed in HEK293S stable cell lines as previously described (15) and purified by immunoaffinity using α‐c‐myc agarose (Thermo Fisher, Waltham, MA, USA). Fibrinogen, purified from human plasma, was purchased from Sigma‐Aldrich (St. Louis, MO, USA). ADAMTS‐13 was pre‐incubated at 37 °C for 1 h in the presence of 5 mm CaCl2 prior to the addition of Fibrinogen to a final concentration of 1 mg mL−1. Reactions were incubated at 37 °C and stopped after 180 min by the addition of SDS PAGE loading buffer. Samples were run on 4–12% BIS‐TRIS gels in MOPS buffer (Invitrogen, Carlsbad, CA, USA) and stained with Coomassie or transferred to nitrocellulose for Western blot using a pAb against the Aα chain (residues 21–320) of human Fibrinogen (Abcam, Cambridge, UK).
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