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A0080

Manufactured by Agilent Technologies
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Sourced in United Kingdom, Denmark

The A0080 is a laboratory instrument designed for analytical measurements. It is a core component for various scientific and industrial applications.

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Lab products found in correlation

A31572, by Thermo Fisher Scientific (2 mentions) Fibrinogen, by Agilent Technologies (1 mentions) 510 confocal microscope, by Zeiss (1 mentions) Apochromat water immersion objectives, by Zeiss (1 mentions) Af385, by R&D Systems (1 mentions) Dylight 488 conjugated l esculentum lectin, by Vector Laboratories (1 mentions) Proteinase k, by Agilent Technologies (1 mentions) Sk 4105, by Vector Laboratories (1 mentions) Bx43 microscope, by Olympus (1 mentions) X0903, by Agilent Technologies (1 mentions) Mp 7451, by Vector Laboratories (1 mentions) Bloxall, by Vector Laboratories (1 mentions) Pp 229 aa, by Biocare Medical (1 mentions) Benchmark ultra, by Roche (1 mentions) S2367, by Agilent Technologies (1 mentions) Dmi8 microscope, by Leica (1 mentions) Target retrieval solution, by Agilent Technologies (1 mentions) H3570, by Thermo Fisher Scientific (1 mentions) Fluorescent mounting medium, by Agilent Technologies (1 mentions) Gelcode blue safe protein stain, by Thermo Fisher Scientific (1 mentions)

14 protocols using a0080

1

Baboon Cerebrovascular Occlusion Protocol

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Postmortem brain tissues were obtained from adult baboons (Papio Anubis) weighing 16–20 Kg (7–12 years old), housed at the Institute of Primate Research (IPR), National Museums of Kenya. The IPR internal review board of the National Museums granted ethical approval and permission for this entire study. As described previously (13), the animals were subjected to permanent occlusion of both the internal carotid arteries and a left vertebral artery for survival periods of 1, 3, 7, 14, 21 and 28 days, as well as sham operated animals (n = 4–8 each group). We evaluated densities of capillary endothelium using H&E and COL4 immunohistochemistry as described above, and the BBB leakage with fibrinogen (1:2000 dilution, A0080, Dako, Cambridge, UK) (13). Pericytes in the deep WM were determined in coronal sections at the level of the frontal lobe in the same manner as described above in human brains. Percentage of fibrinogen stained area (%) in the WM was calculated to evaluate BBB leakage.
Ding R., Hase Y., Ameen‐Ali K.E., Ndung'u M., Stevenson W., Barsby J., Gourlay R., Akinyemi T., Akinyemi R., Uemura M.T., Polvikoski T., Mukaetova‐Ladinska E., Ihara M, & Kalaria R.N. (2020). Loss of capillary pericytes and the blood–brain barrier in white matter in poststroke and vascular dementias and Alzheimer’s disease. Brain Pathology, 30(6), 1087-1101.
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2

Immunofluorescence Analysis of Pericyte Coverage and Fibrin Deposition

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All analyses on human tissue were performed as we previously described61 (link). Heat-induced antigen retrieval was performed following Dako's protocol. For immunofluorescence analysis, we used the following primary antibodies: for pericyte coverage - polyclonal goat anti-human PDGFRβ (R&D systems, AF385; 1:100), for fibrinogen and fibrin extravascular deposits - polyclonal rabbit anti-human fibrinogen (Dako, A0080; 1:500), and species-specific fluorochrome-conjugated secondary antibodies were incubated (see table below) for 1 h at room temperature. Blood vessel endothelial profiles were stained by Dylight 488-conjugated L. esculentum lectin (Vector Labs, DL-1174; 1:200) for 1 h at room temperature. All slices were scanned using Zeiss 510 confocal microscope with Zeiss Apochromat water immersion objectives (Carl Zeiss MicroImaging Inc., Thornwood, NY, USA).
Montagne A., Nikolakopoulou A.M., Zhao Z., Sagare A.P., Si G., Lazic D., Barnes S.R., Daianu M., Ramanathan A., Go A., Lawson E.J., Wang Y., Mack W.J., Thompson P.M., Schneider J.A., Varkey J., Langen R., Mullins E., Jacobs R.E, & Zlokovic B.V. (2018). Pericyte degeneration causes white matter dysfunction in the mouse CNS. Nature medicine, 24(3), 326-337.
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3

Quantifying Fibrin/Fibrinogen in Airway Casts

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After assessment of airway casts by microdissection, immunohistochemical staining for fibrin/fibrinogen was performed on 4-μm paraffin-embedded tissue sections. The embedding orientation of lung lobes allowed for sections to capture cross-sectional views of the location assessed via cast scoring analysis. After deparaffinization, sections were treated with proteinase K (Dako S3020) for 6 min, and endogenous peroxidase activity blocked with BLOXALL® (Vector Labs SP-6000) for 10 minutes. Nonspecific binding was blocked by incubation in 2.5% normal goat serum (Vector Labs MP-7451) for 60 min at room temperature (RT). Fibrin/fibrinogen was detected by incubating in a polyclonal rabbit anti-fibrinogen primary antibody (Dako A0080) diluted 5000-fold in 1% BSA for 60 min at RT, followed by ImmPRESS HRP Polymer goat anti-rabbit IgG Reagent (Vector Labs MP-7451) for 30 min at RT. Sections were developed with DAB (3,3’-Diaminobenzidine; Vector Labs SK-4105), then counterstained with hematoxylin. Rabbit immunoglobulin fraction (normal) (Dako X0903) diluted 21,750-fold in 1% BSA was used as an isotype control. Images were acquired using an Olympus BX43 Microscope with an UPlan FLN 4×/0.13 objective and Olympus cellSens Entry 1.18 software.
Nick H.J., Rioux J.S., Veress L.A., Bratcher P.E., Bloomquist L.A., Anantharam P., Croutch C.R., Tuttle R.S., Peters E., Sosna W, & White C.W. (2020). Alleviation of methyl isocyanate–induced airway obstruction and mortality by tissue plasminogen activator. Annals of the New York Academy of Sciences, 1479(1), 134-147.
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4

Immunohistochemical Analysis of Lung Autopsy

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Conventional hematoxylin and eosin (H&E) staining was performed on deparaffinized sections of the FFPE autopsy asseverates of the lungs according to the current accredited staining protocol at the Institute of Medical Genetics and Pathology of the University Hospital Basel, Switzerland, as per May 2020. Immunohistochemistry was performed according to the current accredited staining protocols, applying the polyclonal ready‐to‐use antibody PP 229 AA from Biocare (Pacheco, CA, USA) against cleaved caspase‐3 on an automated immunostainer Benchmark Ultra (Roche/Ventana, Tucson, AZ, USA), and the polyclonal antibody A0080 from Dako (Glostrup, Denmark) against fibrin(‐ogen) at a dilution of 1:100,000 utilizing detection with a secondary anti‐rabbit link antibody.
Yekelchyk M., Madan E., Wilhelm J., Short K.R., Palma A.M., Liao L., Camacho D., Nkadori E., Winters M.T., Rice E.S., Rolim I., Cruz‐Duarte R., Pelham C.J., Nagane M., Gupta K., Chaudhary S., Braun T., Pillappa R., Parker M.S., Menter T., Matter M., Haslbauer J.D., Tolnay M., Galior K.D., Matkwoskyj K.A., McGregor S.M., Muller L.K., Rakha E.A., Lopez‐Beltran A., Drapkin R., Ackermann M., Fisher P.B., Grossman S.R., Godwin A.K., Kulasinghe A., Martinez I., Marsh C.B., Tang B., Wicha M.S., Won K.J., Tzankov A., Moreno E, & Gogna R. (2021). Flower lose, a cell fitness marker, predicts COVID‐19 prognosis. EMBO Molecular Medicine, 13(11), e13714.
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5

Immunohistochemical Analysis of Thrombi

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Thrombi before in flow injection and thrombi retrieved by EVT from the simulation station were fixed for 48 h in 3.7% paraformaldehyde (PFA), embedded longitudinally in paraffin, and sectioned at 6 μm. After deparaffinization, antigen retrieval with Tris EDTA pH 9.0 (Target Retrieval Solution, S2367, Dako), and blocking for 1 h with 3% of bovine serum albumin (BSA) in PBS 1X, the tissue sections were incubated with primary antibodies to CD42b (2 μg/ml, IM0409, Beckman Coulter), glycophorin A (10 μg/ml, M0819, Dako), and fibrinogen (10 μg/ml, A0080, Dako) for 2 h at room temperature. After being washed two times in PBS, followed by 1 h of incubation with secondary antibodies, tissue sections were counterstained with Hoechst 33,342 (10 μg/ml, H3570, Life Technologies), and mounted in a fluorescent mounting medium (Dako). Hematoxylin and Eosin (H&E) staining was also performed as described (13 (link)). The H and E and fluorescent images were acquired using a Hamamatsu (Japan) nanozoomer slide scanner and a Leica DMi8 microscope, respectively (Leica Microsystems, Wetzlar, Germany), with LAS X Software.
Freiherr von Seckendorff A., Delvoye F., Levant P., Solo Nomenjanahary M., Ollivier V., Bourrienne M.C., Di Meglio L., Piotin M., Escalard S., Maier B., Hebert S., Smajda S., Redjem H., Mazighi M., Blanc R., Ho-Tin-Noé B, & Désilles J.P. (2022). Modeling Large Vessel Occlusion Stroke for the Evaluation of Endovascular Therapy According to Thrombus Composition. Frontiers in Neurology, 12, 815814.
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6

Fibrin Clot Protein Analysis

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Clots were made using plasma by the addition of thrombin (0.5 U/ml) and CaCl2 (5 mM), with and without FXIII inhibitor T101 (1 mM). The fibrin film was removed from each clot and reduced by the addition of NuPAGE sample reducing agent (100 mM DTT) and heating at 95°C for 15 minutes. Fibrin samples were prepared by the formation of a clot with IF-1 fibrinogen (1 mg/ml), addition of thrombin and CaCl2, and reduction as described above. FXIII-, BSA-, and IF-1–purified fibrinogen samples were reduced in a similar way and run alongside the films to help identify bands in the gel, and as controls for the blots. Protein concentrations were determined using NanoDrop to load 2 μg of each protein sample on 2 identical 4%–12% NuPAGE Bis-Tris gels. After running, one gel was stained using GelCode Blue Safe Protein Stain (Thermo Fisher Scientific), and one was transferred to a PVDF membrane (Thermo Fisher Scientific). The membrane was blocked overnight using 4% skim milk in 50 mM Tris, 150 mM NaCl, 0.1% Tween-20. Polyclonal rabbit anti–human fibrinogen antibody (A0080; Dako) was added to the blot in blocking buffer and detected using goat anti-rabbit HRP secondary antibody (P0448; Dako). Signal was detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).
Macrae F.L., Duval C., Papareddy P., Baker S.R., Yuldasheva N., Kearney K.J., McPherson H.R., Asquith N., Konings J., Casini A., Degen J.L., Connell S.D., Philippou H., Wolberg A.S., Herwald H, & Ariëns R.A. (2018). A fibrin biofilm covers blood clots and protects from microbial invasion. The Journal of Clinical Investigation, 128(8), 3356-3368.
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7

Immunohistochemical Identification of Muscle Fiber Types

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For immunohistochemical (IHC) analyses, monoclonal mouse anti-myosin Skeletal fast M4276 and Skeletal slow M8421 antibodies (Abs) (Sigma Co., St. Louis, MO, USA) were utilized for demonstration of the fast and slow myosin heavy chain (MHC) isoforms, type II and type I, respectively. Polyclonal rabbit anti-human myoglobin (A0324) and anti-human fibrinogen (A0080) Abs (Dako, Glostrup, Denmark) were employed for Mb and fibrinogen. IHC visualization was achieved by the avidin-biotin-peroxidase method (Vector Laboratories, Burlingame, California, USA). Tissue sections in which the primary Abs were replaced by phosphate-buffered saline or nonimmune serum (rabbit or mouse) were used as negative controls to confirm the specificity of the test and no immunolabelling was observed44 (link).Goat and human tissues were used as positive controls. Slow MHC recognises type I fibres (slow-twitch fibres) and fast MHC recognises type IIa, IIb and IId (IIx) (in fast-twitch fibres). Immunohistochemical identification of muscle fibre types was performed on 15 different species.
Sierra E., Fernández A., Espinosa de los Monteros A., Díaz-Delgado J., Bernaldo de Quirós Y., García-Álvarez N., Arbelo M, & Herráez P. (2015). Comparative histology of muscle in free ranging cetaceans: shallow versus deep diving species. Scientific Reports, 5, 15909.
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8

Multiplex Immunofluorescence for Tumor Microenvironment

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Primary antibodies: PyMT-antibody (ab15085, Abcam), anti-CD31 (ab24590, Abcam), anti-Ki67 (ab15580, Abcam), anti-cleaved caspase-3 (9661, Cell Signaling), anti-F4/80 Alexa Fluor 488 conjugate (MF48020, Life Technologies), anti-human fibrinogen (A0080, Dako), E-cadherin (sc-7870, Santacruz), CCL2/MCP-1 (sc-28879, Santacruz), anti-HGF (AF2207, R&D systems), anti-beta actin (BA3R, Thermo Scientific). Secondary antibodies: goat anti-mouse Alexa Fluor 488 (A11017, Invitrogen), goat anti-rabbit Alexa Fluor 594 (A11012, Invitrogen), goat anti-rat Alexa Fluor 680 (A21096, Invitrogen), goat anti-rat Alexa Fluor 568 (A11077, Invitrogen), donkey anti-rabbit Alexa Fluor 555 (A31572, Invitrogen), anti rabbit IR-Dye 800CW, anti goat IR-Dye 680CW, anti-mouse IRDye 800CW, anti-mouse IRDye 680CW
Roy A., Femel J., Huijbers E.J., Spillmann D., Larsson E., Ringvall M., Olsson A.K, & Åbrink M. (2016). Targeting Serglycin Prevents Metastasis in Murine Mammary Carcinoma. PLoS ONE, 11(5), e0156151.
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9

Adipose and Liver Histological Analysis

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Formalin-fixed, paraffin-embedded sections of adipose and liver were stained with either H & E or mason’s trichrome stain. For fibrin immunohistology, AT sections were incubated overnight at 4οC with the primary antibody, polyclonal rabbit anti-human fibrinogen (A0080 from Dako), The slides were washed and treated sequentially with biotinylated goat anti-rat IgG (Jackson Immunoresearch), streptavidin-peroxidase conjugate (Zymed), and diaminobenzidine chromogen containing 0.03% hydrogen peroxide (Vector Laboratories), counterstained with Gill modified hematoxylin, and mounted in GVA-mount (Zymed). Immunofluorescence staining for Plg-RKT in human and mouse AT was done using the pan specific primary antibody mouse anti- PlgRKT mAb 7H1 previously described (38 (link)). Human adipose macrophages were stained using rabbit anti-CD80 Ab (Santa Cruz Biotechnology) and mouse ATM stained using rat anti-F4/80 Ab (ThermoFisher). Quantitative analysis of immunohistochemical data was done using QuPath v0.3.0.
Samad F., Bai H., Baik N., Haider P., Zhang Y., Rega-Kaun G., Kaun C., Prager M., Wojta J., Bui Q., Chakrabarty S., Wang J., Parmer R.J, & Miles L.A. (2021). The plasminogen receptor Plg-RKT regulates adipose function and metabolic homeostasis. Journal of thrombosis and haemostasis : JTH, 20(3), 742-754.
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10

Histological Analysis of Murine Feet

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Fixed murine feet were decalcified using the EDTA-based Osteosoft solution (Merck) and then embedded in paraffin for histological analysis.by Ziehl-Neelsen stain, Alcian blue-periodic acid Schiff stain, and immunohistochemistry (IHC) for fibrin(ogen). For IHC staining, 5-μm tissue sections on polylysine-coated slides were deparaffinised, endogenous peroxidase quenched, epitope unmasked with heated IHC citrate buffer (pH 6.0) (Merck) and blocked with 5% bovine serum albumin. The tissue sections were incubated with anti-fibrinogen antibody (A0080, DAKO) or matched isotype control overnight at 4°C. Staining was then performed with biotinylated horse anti-rabbit IgG (Vector Laboratories) and VECTASTAIN Elite ABC kit and ImmPACT NovaRED peroxidase substrate and further counterstained with Harris Haematoxylin (ThermoFisher Scientific). Whole slide images were captured using the NanoZoomer slide scanner (Hamamatsu Photonics) and analysed using ImageScope software (Leica Biosystems) and ndp2.view software (Hamamatsu). Some photographs were taken with Micropix microscope camera (acquisition software Cytocam) attached to a Yenway CX40 laboratory microscope (Micropix).
Hsieh L.T., Hall B.S., Newcombe J., Mendum T.A., Umrania Y., Deery M.J., Shi W.Q., Salguero F.J, & Simmonds R.E. (2023). Mycolactone causes catastrophic Sec61-dependent loss of the endothelial glycocalyx and basement membrane: a new indirect mechanism driving tissue necrosis in Mycobacterium ulcerans infection. bioRxiv.
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