A0262

Manufactured by Agilent Technologies

Sourced in Denmark

The A0262 is a laboratory instrument designed for general analytical applications. It provides accurate and reliable measurements within its intended functional parameters.

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Lab products found in correlation

P0217, by Agilent Technologies (1 mentions) Fluorescein isothiocyanate (fitc), by Jackson ImmunoResearch (1 mentions) Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions) Alexa fluor 488 conjugated anti rat igg antibody, by Thermo Fisher Scientific (1 mentions) Sola15, by Thermo Fisher Scientific (1 mentions) Eclipse ni e microscope, by Nikon (1 mentions) Alexa fluor 546 conjugated anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions) Fluorsave reagent, by Merck Group (1 mentions)

4 protocols using a0262

IgA and IgM B Cell Culture and Quantification

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Cells were flow sorted as IgA⁺ (n = 5) or IgM⁻ (n = 3) and cultured for 40 h in RPMI medium with 10% heat-inactivated fetal calf serum, l-glutamine, and penicillin-streptomycin at a density of 2.5 × 10⁵ cells/ml before cells were spun down at 500 rcf/7 min, and the supernatant was transferred in twofold dilution to 96-well plates precoated with rabbit anti–human IgA (A0262; Dako) and blocked with 1% BSA. Purified IgA monomer at known concentration was used as standard (courtesy of R. Iversen, Oslo University Hospital-Rikshospitalet and the University of Oslo, Oslo, Norway). Bound IgA was detected with goat anti–human IgA peroxidase conjugate (A0295; Sigma-Aldrich) and tetramethylbenzidine (Kirkegaard & Perry Laboratories).

Landsverk O.J., Snir O., Casado R.B., Richter L., Mold J.E., Réu P., Horneland R., Paulsen V., Yaqub S., Aandahl E.M., Øyen O.M., Thorarensen H.S., Salehpour M., Possnert G., Frisén J., Sollid L.M., Baekkevold E.S, & Jahnsen F.L. (2017). Antibody-secreting plasma cells persist for decades in human intestine. The Journal of Experimental Medicine, 214(2), 309-317.

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IgA Protease Cleavage Assay

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A subset of 54 isolates, representing all IgA protease gene variants, was chosen for confirmation of IgA1 cleavage by Western blot. The strains were grown in brain-heart-infusion (BHI) broth supplemented with 10 μg/ml NAD and hemin at 37 °C 5% CO₂ overnight. The bacteria were spun down and 20 μl of supernatant was incubated with 3 μl of 1 mg/ml human IgA1 (Calbiochem, Solna, Sweden) at 37 °C overnight. The proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with polyclonal rabbit anti-human IgA antibodies at dilution 1:1000 overnight at 4 °C (A0262, Dako; Glostrup Denmark) followed by polyclonal HRP (horseradish peroxidase)-conjugated swine anti-rabbit IgG (dilution 1:1000) (P0217, Dako; Glostrup, Denmark) 2 h at room temperature and developed.

Resman F., Manat G., Lindh V., Murphy T.F, & Riesbeck K. (2018). Differential distribution of IgA-protease genotypes in mucosal and invasive isolates of Haemophilus influenzae in Sweden. BMC Infectious Diseases, 18, 592.

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Dual Immunofluorescent Staining of Mucosal Markers

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Paraffin sections (5 μm) were cut from each tissue block. Two serial sections from each tissue block were stained with hematoxylin and eosin (H&E) for routine histologic evaluation or the periodic acid-Schiff (PAS) reaction for detection of mucus. Double immunofluorescent immunohistochemistry (IHC) was performed using two different pairs of primary antibodies – IgA and pIgR or MUC5AC and MUC5B. Primary antibodies used were rabbit polyclonal for IgA (A0262, DakoCytomation, Carpinteria, CA) and MUC5B (ABIN739920, Antibodies-online, Atlanta, GA) or murine monoclonal for pIgR (clone SC-05, Abcam Inc., Cambridge, MA) and MUC5AC (clone CLH2, MAB2011, EMD Millipore Corporation, Billerica, MA). Secondary anti-rabbit or anti-mouse antibodies were labeled with FITC or Cy3, respectively (Jackson ImmunoResearch Laboratory). The murine monoclonal anti-pIgR antibody demonstrated consistent staining on the basolateral surfaces rather than the apical surfaces of ciliated epithelial cells, suggesting that the antibody binds only pIgR, and not the SC of SIgA molecules. The specificity of IHC was verified using an antibody isotype control replacing the primary antiserum with an identical concentration of non-immunized mouse or rabbit serum (Invitrogen Corporation, Camarillo, CA).

Du R.H., Richmond B., Blackwell TS J.r., Cates J.M., Massion P.P., Ware L.B., Lee J.W., Kononov A.V., Lawson W.E., Blackwell T.S, & Polosukhin V.V. (2015). Secretory IgA from submucosal glands does not compensate for its airway surface deficiency in chronic obstructive pulmonary disease. Virchows Archiv : an international journal of pathology, 467(6), 657-665.

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Immunofluorescence Staining of Colitis

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Immunofluorescence staining was performed on five patients with untreated active colitis and five HCs. Formalin-fixed, paraffin-embedded human tissue sections 3 μm in thickness were treated in xylene, a decreasing alcohol gradient and distilled water to achieve de-waxing and rehydration of the tissue. Heat-induced epitope retrieval was performed for 15 min in Tris-EDTA buffer (pH 9). After epitope retrieval, tissue sections were permeabilized with 0.2% Triton X-100 in PBS, blocked with 5% BSA and 5% Fc receptor blocking reagant (Miltenyi Biotec) and stained with polyclonal rabbit anti-human IgA (A0262, DAKO, 1:1,000 dilution), monoclonal mouse anti-human CD79a (clone JCB117, DAKO, 1:25 dilution) and monoclonal rat anti-human Ki67 (SolA15, Invitrogen, 1:50 dilution). Staining with primary antibodies was followed by detection with polyclonal donkey Alexa Fluor 546-conjugated anti-mouse IgG antibody (Invitrogen, 1:200), Alexa Fluor 647-conjugated anti-rabbit IgG antibody (Invitrogen, 1:500) and Alexa Fluor 488-conjugated anti-rat IgG antibody (Invitrogen, 1:500). Nuclear DNA was visualized with DAPI, and coverslips were applied with FluorSave reagent (Merck Millipore). Images were acquired with either a Leica TCS SP5 upright confocal microscope or a Nikon Eclipse Ni-E microscope and were further analyzed with ImageJ software.

Uzzan M., Martin J.C., Mesin L., Livanos A.E., Castro-Dopico T., Huang R., Petralia F., Magri G., Kumar S., Zhao Q., Rosenstein A.K., Tokuyama M., Sharma K., Ungaro R., Kosoy R., Jha D., Fischer J., Singh H., Keir M.E., Ramamoorthi N., O’ Gorman W.E., Cohen B.L., Rahman A., Cossarini F., Seki A., Leyre L., Vaquero S.T., Gurunathan S., Grasset E.K., Losic B., Dubinsky M., Greenstein A.J., Gottlieb Z., Legnani P., George J., Irizar H., Stojmirovic A., Brodmerkel C., Kasarkis A., Sands B.E., Furtado G., Lira S.A., Tuong Z.K., Ko H.M., Cerutti A., Elson C.O., Clatworthy M.R., Merad M., Suárez-Fariñas M., Argmann C., Hackney J.A., Victora G.D., Randolph G.J., Kenigsberg E., Colombel J.F, & Mehandru S. (2022). Ulcerative colitis is characterized by a plasmablast-skewed humoral response associated with disease activity. Nature medicine, 28(4), 766-779.

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