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M0814

Manufactured by Agilent Technologies
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Sourced in Denmark, United States

The M0814 is a laboratory instrument designed for analytical testing and measurement applications. It is a compact and versatile device that can perform various analytical tasks. The core function of the M0814 is to provide accurate and reliable data, enabling users to conduct their experiments and analyses with confidence. Further details on the intended use or specific applications of this product are not available.

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Lab products found in correlation

A0398, by Agilent Technologies (3 mentions) M0823, by Agilent Technologies (2 mentions) M7103, by Agilent Technologies (2 mentions) A0452, by Agilent Technologies (2 mentions) M0755, by Agilent Technologies (2 mentions) Ab64693, by Abcam (2 mentions) P0260, by Agilent Technologies (1 mentions) X0943, by Agilent Technologies (1 mentions) Nanozoomer slide scanner, by Hamamatsu Photonics (1 mentions) Vimentin, by Santa Cruz Biotechnology (1 mentions) Sc 6260, by Santa Cruz Biotechnology (1 mentions) Myogenin, by Santa Cruz Biotechnology (1 mentions) Hoechst 33258, by Thermo Fisher Scientific (1 mentions) Ab15580, by Abcam (1 mentions) Sc 304, by Santa Cruz Biotechnology (1 mentions) Alexa fluor 594 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions) Cd3 2gv6, by Roche (1 mentions) Ls c96142, by LSBio (1 mentions) Alexafluor 488 conjugated goat anti rabbit antibodies, by Thermo Fisher Scientific (1 mentions) L9393, by Merck Group (1 mentions)

26 protocols using m0814

1

Immunohistochemical Analysis of PLTP Expression in Synovial Tissue

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Synovial tissues were isolated from patients who were undergoing total joint replacement surgery or synovectomy. Briefly, tissues were fixed in formalin and embedded in paraffin. Immunochemistry was performed after deparaffinization, rehydratation, and unmasking by heating in 10 mmol/L sodium citrate buffer (pH 6). Unspecific sites were saturated with PBS containing 10% horse serum and endogenous peroxidase activity was blocked using 3% hydrogen peroxide. Sections were then incubated with a mouse monoclonal antibody to PLTP (5μg/ml; ab57273; Abcam) or mouse anti-CD68 antibody (2.5 μg/ml; M0814; Dako). HRP-coupled secondary antibodies were added and incubated at room temperature for 1 hour. Visualization of immunohistochemical section was performed using Nanozoomer slide scanner (Hamamatsu Photonics). Double staining using antibodies specific for macrophages (mouse anti-CD68 antibody (2.5 μg/ml; M0814; Dako) or RA-FLS (mouse anti-alpha-smooth muscle Actin (α-SMA) (1 μg/ml; 1A4; eBioscience) and PLTP (rabbit anti-PLTP antibody (1/200- NB400-106; Novus Biological) was also performed. As a negative control, sections were incubated with isotype control antibodies (mouse IgG MOPC21 (M7894-Sigma) or rabbit IgG (X0936-Jackson Immunoresearch). Visualization of immunofluorescence in synovial section was performed using Leica DM 6000 microscope (Leica Microsytems).
Audo R., Deckert V., Daien C.I., Che H., Elhmioui J., Lemaire S., Pais de Barros J.P., Desrumaux C., Combe B., Hahne M., Lagrost L, & Morel J. (2018). PhosphoLipid transfer protein (PLTP) exerts a direct pro-inflammatory effect on rheumatoid arthritis (RA) fibroblasts-like-synoviocytes (FLS) independently of its lipid transfer activity. PLoS ONE, 13(3), e0193815.
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2

Histological Analysis of Aneurysm Walls

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Aneurysm wall specimens were collected at surgery (clipping) for histological study when available. Routine histological analysis was performed. Formalin fixed and paraffin embedded specimens were cut into 2.5 μm thick tissue sections. Slides were stained with hematoxylin–eosin. Immunohistochemical staining was performed. The following primary antibodies were used: CD68 (M0814; Dako, Glostrup, Denmark) as an inflammatory marker and CD31 (M0823; Dako) to assess neoangiogenesis.
Liu X., Feng J., Li Z., Zhang Z., Zhang Q., Jiang Y., Huo X., Chai X., Wu Y., Kong Q., Liu P., Ge H., Jin H., An J., Jiang P., Saloner D.A., Li Y, & Zhu C. (2021). Quantitative analysis of unruptured intracranial aneurysm wall thickness and enhancement using 7T high resolution, black blood magnetic resonance imaging. Journal of neurointerventional surgery, 14(7), 723-728.
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3

Immunohistochemical Analysis of Micromasses

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For immunohistochemical analysis, micromasses were fixated for 2 h in 2 % paraformaldehyde in phosphate-buffered saline (PBS)/1 mM CaCl2, dehydrated and embedded in paraffin. Sections 7-μm thick were deparaffinized, rehydrated and incubated with antibodies 11-fibrau (1:100 for 60 minutes) (clone D7-fib, Imgen, distributed by ITK Diagnostics, Uithoorn, The Netherlands), mouse anti-human cluster of differentiation 68 (CD68) (1:100 for 60 minutes) (M0814, DAKO, Heverlee, Belgium) or control mouse IgG2ak (X0943, DAKO) and IgG1k (X0931, DAKO) respectively. Endogenous peroxidase activity was blocked with 3 % H2O2 (Merck Millipore, Amsterdam, The Netherlands) in methanol. Subsequently, the sections were incubated with the secondary horseradish peroxidase (HRP)-conjugated rabbit-anti-mouse IgA/G/M (1:200 for 60 minutes) (P0260, DAKO). Peroxidase was developed with diaminobenzidine and counterstained with hematoxylin for 60 seconds.
Broeren M.G., de Vries M., Bennink M.B., Arntz O.J., van Lent P.L., van der Kraan P.M., van den Berg W.B., van den Hoogen F.H., Koenders M.I, & van de Loo F.A. (2016). Suppression of the inflammatory response by disease-inducible interleukin-10 gene therapy in a three-dimensional micromass model of the human synovial membrane. Arthritis Research & Therapy, 18, 186.
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4

Immunohistochemistry and Immunofluorescence Assay

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ALOX5 (Abcam #ab169755; IHC and IF: 1:100), CD68 (DAKO M0814 [RRID:AB_2314148]; IHC: 1:200; IF: 1:100), human leukocyte antigen G (HLAG) (BD Pharmingen cat#557577 [RRID:AB_396753]; IHC: 1:500; IF: 1:200), PFN1 (Santa Cruz Biotech #sc-137236 [RRID:AB_2163203]; IHC 1:400), vimentin (Santa Cruz Biotech #sc-6260 [RRID:AB_628437]; IF: 1:100). Negative control isotype rabbit, goat or mouse IgG (Dako) was included for every tissue section.
Menkhorst E.M., Van Sinderen M.L., Rainczuk K., Cuman C., Winship A, & Dimitriadis E. (2017). Invasive trophoblast promote stromal fibroblast decidualization via Profilin 1 and ALOX5. Scientific Reports, 7, 8690.
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5

Immunofluorescent Characterization of Muscle Biopsies

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5–10 μm cryosections of patient and control biopsies were stored until analysis at −80 °C. Sections were thawed before staining. Primary antibodies were directed against CD68, clone KP1 (M0814; DAKO; 1:800), eMyHC (F1.652; DSHB; 1:150), Ki67 (Ab15580; Abcam; 1:50), Laminin (L9393; Sigma; 1:500 or LS-C96142; LS Bio; 1:500), MyoD (SC304; Santa Cruz; 1:200), Myogenin (M-225 ; Santa Cruz; 1:200), Pax7 (DSHB; 1:50), CD3 (2GV6; Ventana; ready to use), CD20Cy; clone L26; Dako, 1:400). Following the primary antibody incubation, sections were rinsed and incubated with biotin-conjugated anti-mouse antibody (BA-2000; Vector labs,1:50) and then with Alexafluor594-conjugated streptavidin (S11227; Life Technologies, 1:500). Sections incubated with rabbit primary antibodies were subsequently incubated with Alexafluor 488-conjugated goat-anti rabbit antibodies (A11307; Life Technologies,1:500). Finally chicken anti-Laminin was detected with Alexafluor647-conjugated goat anti-chicken antibodies (A21449; Life Technologies, 1:500). All sections were counterstained with Hoechst33258 (H3569; Life Technologies, 1:15000). The slides were mounted with Mowiol (475904; Calbiochem). For some biopsies, limited amounts of sections of sufficient quality were available for immunofluorescent analysis, and not all stainings could be performed for all patients.
Schaaf G.J., van Gestel T.J., Brusse E., Verdijk R.M., de Coo I.F., van Doorn P.A., van der Ploeg A.T, & Pijnappel W.P. (2015). Lack of robust satellite cell activation and muscle regeneration during the progression of Pompe disease. Acta Neuropathologica Communications, 3, 65.
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6

Immunohistochemical Profiling of Tissue Samples

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Slides were de-paraffinized in xylene and rehydrated through graded ethanol followed by heat mediated antigen retrieval using 10 mM Sodium Citrate Buffer (pH 6.0). IHC was performed via incubations with one primary antibody followed by host-matched secondary polymer reagent and color substrate. Primary antibodies used were as follows: CD3+, CD8+, HLA-DR, CD68 (A0452, M7103, M0746, M0814 – Dako), CD163, Ki-67, and αSMA (ab189915, ab16667, ab5694 – Abcam), cleaved-Caspase-3 (#9661, Cell Signal Technologies), and FOXP3 (14–4777–82, eBioscience). Secondary reagents were ImmPress Rabbit HRP and Mouse HRP (Vector Laboratories). Color development was performed using Quanto DAB brown (Fisher Scientific). Slides were counterstained with Harris hematoxylin (Sigma) as appropriate, dehydrated and mounted.
Jiang X., Seo Y.D., Chang J.H., Coveler A., Nigjeh E.N., Pan S., Jalikis F., Yeung R.S., Crispe I.N, & Pillarisetty V.G. (2017). Long-lived pancreatic ductal adenocarcinoma slice cultures enable precise study of the immune microenvironment. Oncoimmunology, 6(7), e1333210.
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7

Synovial Tissue Immunofluorescence Staining

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Double fluorescent labelling of MS4A7 (HPA017418, Sigma, dilution 1:50) and CD68 (M0814, dilution 1:50; Dako) was performed on synovial sections by multiplex immunofluorescence staining using a tyramide signal amplification protocol (Invitrogen, Thermo Fisher Scientific). All the slides were counterstained with DAPI (Invitrogen, Thermo Fisher Scientific) and digitally scanned using Nanozoomer S60 (Hamamatsu Photonics).
Silva‐Gomes R., Mapelli S.N., Boutet M., Mattiola I., Sironi M., Grizzi F., Colombo F., Supino D., Carnevale S., Pasqualini F., Stravalaci M., Porte R., Gianatti A., Pitzalis C., Locati M., Oliveira M.J., Bottazzi B, & Mantovani A. (2021). Differential expression and regulation of MS4A family members in myeloid cells in physiological and pathological conditions. Journal of Leukocyte Biology, 111(4), 817-836.
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8

Quantitative Immune Cell Profiling

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Quantification of immune cells (T cells, cytotoxic T cells, macrophages, plasma cells, and neutrophils) was assessed by IHC staining on tissue microarrays and automatic quantification by QuPath63 (link). CD45Ro (Ventana; 790-2930; Mouse monoclonal (UCHL-1)) for total immune cells, CD3 (Ventana; 790-4341; Rabbit monoclonal (2GV6)) for total T cells, CD8 (NOVO; PA0183; Mouse monoclonal (4B11)) for cytotoxic T cells, Foxp3 (Abcam; ab20034; Mouse monoclonal (236A/E7)) for Tregs, CD68 (DAKO; M0814; Mouse monoclonal (KP1)) for macrophages, CD163 (NOVO; NCL-CD163; Mouse monoclonal (10D6)) for M2 macrophages, MUM1 (DAKO; M7259; Mouse monoclonal (MUM1P)) for plasma cells, and MPO (DAKO; A0398; Rabbit polyclonal) for neutrophils were used as cell type markers. Four-μm-thick glass slides were stained using Ventana BenchMark XT and OptiView universal DAB staining kit (Ventana #760-700). Staining procedure of automatic stainer is antigen retrieval (100 °C for 24 min in citrate buffer), peroxidase inhibition (37 °C for 4 min in 3% H2O2), primary antibody (37 °C for 16 min), Linker (HQ linker; 37 °C for 8 min), polymer amplification (HRP multimer; 37 °C for 8 min), chromogen by DAB (37 °C for 8 min), counterstaining by hematoxylin (37 °C for 8 min), and post counterstain (37 °C for 4 min).
Yoon S.H., Choi S.W., Nam S.W., Lee K.B, & Nam J.W. (2021). Preoperative immune landscape predisposes adverse outcomes in hepatocellular carcinoma patients with liver transplantation. NPJ Precision Oncology, 5, 27.
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9

Fetuin-A Detection in Activated Microglia

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This staining was performed to detect fetuin-A in activated microglia. In brief: CD68 was stained using a monoclonal mouse-anti-human antibody (Dako Cat# M0814, RRID:AB_2314148, clone KP1, dilution 1:50) and a polyclonal goat-anti-mouse Alexa Fluor 488 conjugated secondary antibody (Thermo Fisher Scientific Cat# A-11029, RRID:AB_2534088, dilution 1:300). Fetuin-A was detected as described above. Staining with Sudan Black and DAPI, washing, mounting and storing was performed as above. For details see http://dx.doi.org/10.17504/protocols.io.syfeftn [PROTOCOL DOI].
Heinen M.C., Babler A., Weis J., Elsas J., Nolte K., Kipp M., Jahnen-Dechent W, & Häusler M. (2018). Fetuin-A protein distribution in mature inflamed and ischemic brain tissue. PLoS ONE, 13(11), e0206597.
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10

Immunohistochemical Staining of FFPE Tissues

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Immunohistochemical (IHC) staining was performed after chemical dewaxing of 4 μm thick formalin-fixed paraffin-embedded (FFPE) tissue sections as described before (30 (link), 34 (link)) using antibodies for CD14 (diluted at 1:50 in PSA) (Chemicon, CA, USA), CD68 (diluted at 1:50 in PSA) (M0814) from Dako (Agilent, CA, USA), CD163 (1:500) (Abcam, Cambridge, UK), and MAC387 (1µg/ml) (Abcam, Cambridge, UK). IHC Staining was carried out by adding 100 µl of DAB+ chromogen diluted at 1:50 in substrate buffer [(EnVision+ Dual Link System-HRP (DAB+)] for 10 min. Finally, tissue specimens were washed in phosphate buffer saline (PBS), the nuclei were counterstained with hematoxylin and mounted using Permount® (Fisher Scientific, PA, USA) for microscopic examination. Negative control slides were run in parallel with each marker where primary antibody was replaced by PBS. The stained area fractions were calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA) (7 (link), 8 (link)).
Mohamed H.T., El-Sharkawy A.A., El-Shinawi M., Schneider R.J, & Mohamed M.M. (2022). Inflammatory Breast Cancer: The Secretome of HCMV+ Tumor-Associated Macrophages Enhances Proliferation, Invasion, Colony Formation, and Expression of Cancer Stem Cell Markers. Frontiers in Oncology, 12, 899622.
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