Mec13

Manufactured by BD

Sourced in United States

The MEC13.3 is a laboratory equipment product manufactured by BD. It is designed to perform a core function, but a detailed and unbiased description is not available at this time.

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Lab products found in correlation

Lsm 710 confocal microscope, by Zeiss (6 mentions) Mwreg30, by BD (5 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions) Collagenase 1, by Thermo Fisher Scientific (3 mentions) Ecgm2, by PromoCell (3 mentions) Anti rat immunoglobulin g coated magnetic beads, by Thermo Fisher Scientific (3 mentions) Dynal mpc s, by Thermo Fisher Scientific (3 mentions) Bovine serum albumin (bsa), by Merck Group (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions) Anti α smooth muscle actin antibody 1a4, by Merck Group (2 mentions) Egm 2 media, by Lonza (2 mentions) Vectastain abc kit, by Vector Laboratories (2 mentions) 3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions) Goat anti rat alexa fluor 555, by Abcam (2 mentions) Axiovision software, by Zeiss (2 mentions) Moflo xdp, by Beckman Coulter (2 mentions) 7 aminoactinomycin d 7 aad, by Thermo Fisher Scientific (2 mentions) Hematoxylin, by Agilent Technologies (2 mentions) Dab liquid system, by Agilent Technologies (2 mentions) Ab4710, by Abcam (2 mentions)

72 protocols using mec13

Antibody-mediated Molecular Targeting

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Antibodies against mouse ICAM-1, PECAM-1, or VCAM-1 were clone YN1 (ATCC; Manassas, VA), MEC13 (BD Biosciences; San Jose, CA), and MK2 (Santa Cruz Biotechnology; Dallas, TX). Non-specific IgG was from Jackson Immunoresearch (Pike West Grove, PA). Recombinant human acid sphingomyelinase (ASM) was provided by Dr. Edward Schuchman (Mount Sinai School of Medicine) [39 (link)]. Green Fluoresbrite Polystyrene particles were from Polysciences (Warrington, PA). Unless otherwise stated, all other reagents were from Sigma Chemical (St. Louis, MO).

Papademetriou I., Tsinas Z., Hsu J, & Muro S. (2014). Combination-targeting to multiple endothelial cell adhesion molecules modulates binding, endocytosis, and in vivo biodistribution of drug nanocarriers and their therapeutic cargoes. Journal of controlled release : official journal of the Controlled Release Society, 188, 87-98.

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Quantifying Tumor Proliferation and Angiogenesis

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Tumors from control and Met-treated animals were excised and placed in fixative for immunostaining and histology. Tumor proliferation index was estimated using Ki-67 stained specimens (mAb; Dako - M7240). Five random areas were captured at 40 × magnification and analyzed using NIH Image J software (NIH, Maryland). Staining intensity was categorized into 1 = negative/no stain, 2 = weak, 3 = moderate and 4 = strong. For each image, 100 cells with nuclei were assessed for staining intensity and assigned a value from 1 to 4. The scores were assessed by an observer who was blinded to the identity of the samples. The H-scores for Ki-67 was calculated as the sum scores of all the 500 cells per sample. Microvessel density was estimated by counting CD31 (MEC13; BD Pharmingen – 550274) positive endothelial clusters with a visible lumen on stained tissue sections. Stained slides were scanned and digitized using Scanscope XT (Aperio Technologies, Vista, CA).

Verma A., Rich L.J., King V.C, & Seshadri M. (2018). Visualizing the Effects of Metformin on Tumor Growth, Vascularity and Metabolism in Head and Neck Cancer. Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology, 47(5), 484-491.

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Immunofluorescence and Flow Cytometry Analysis

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Cells were detached from plates using accutase (Sigma) and fixed in 7.4% formaldehyde in N2B27 media for 10 min. Permeabilisation was carried out using ice-cold methanol, and cells were blocked using 1% BSA. The primary antibody (pS6^240/244 #5264, cleaved caspase-3 #9664, both Cell Signaling; 1:200 dilution) was incubated with cells for 1 h at room temperature. After washing, the secondary antibody was applied (Alexafluor-546/405, Thermo Fisher Scientific; 1:2000 dilution) for 30 min. CD31 (BD Biosciences, MEC13.3; 1:200 dilution) staining was performed on live cells. The antibody was incubated with cells for 45 min at 4 °C and cells were washed in PBS prior to flow cytometry analysis. All flow cytometry was performed using a BD LSR II flow cytometer and analysed using FlowJo software (both BD Biosciences).

Bowling S., Di Gregorio A., Sancho M., Pozzi S., Aarts M., Signore M., D. Schneider M., Martinez-Barbera J.P., Gil J, & Rodríguez T.A. (2018). P53 and mTOR signalling determine fitness selection through cell competition during early mouse embryonic development. Nature Communications, 9, 1763.

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Immunohistochemical and Whole-Mount Staining of Embryos

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The embryos for immunohistochemical and whole-mount staining were fixed in 4% paraformaldehyde. Embryos for immunohistochemical staining were embedded in paraffin and cut into 6- to 7-μm thick sections. Tissues for frozen sections were embedded in OCT (Tissue-Tek) and cut into 8-μm thick sections. The frozen sections were fixed in acetone and acetone-chloroform. The hematoxylin and eosin staining procedure followed standard protocol. The immunohistochemical and immunofluorescent staining with anti-α-smooth muscle actin antibody (1A4, Sigma, 1:400 dilution) or anti-CD31 antibody (MEC13.3, BD, 1:400 dilution) followed basic immunoperoxidase procedures or immunostaining procedure for fluorophore-conjugated secondary antibodies. The procedures for whole-mount anti-CD31 staining were described previously^{4 (link)}.

Fisher O.S., Deng H., Liu D., Zhang Y., Wei R., Deng Y., Zhang F., Louvi A., Turk B.E., Boggon T.J, & Su B. (2015). Structure and vascular function of MEKK3–cerebral cavernous malformations 2 complex. Nature Communications, 6, 7937.

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Embryonic Tissue Staining Protocols

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The embryos for immunohistochemical and whole-mount staining were fixed in 4% paraformaldehyde. Embryos for immunohistochemical staining were embedded in paraffin and cut into 6- to 7-μm thick sections. Tissues for frozen sections were embedded in OCT (Tissue-Tek) and cut into 8-μm thick sections. The frozen sections were fixed in acetone and acetone–chloroform. The haematoxylin and eosin staining procedure followed standard protocol. The immunohistochemical and immunofluorescent staining with anti-α-smooth muscle actin antibody (1A4, Sigma, 1:400 dilution) or anti-CD31 antibody (MEC13.3, BD, 1:400 dilution) followed basic immunoperoxidase procedures or immunostaining procedure for fluorophore-conjugated secondary antibodies. The procedures for whole-mount anti-CD31 staining were described previously4 (link).

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Immunostaining of Mouse Eye Tissues

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Mouse eyes were collected and fixed in 4% paraformaldehyde for 1 hour. Dissected choroid or frozen section was permeabilized in PBS containing 5% normal rabbit serum albumin, 2% bovine serum albumin, and 0·3% Triton X-100 for 1 hour. The following antibodies were used: isolectin IB4 (ThermoFisher Scientific, I21411, RRID: AB_23146), IBA1 (Wako, 019-19741, RRID: AB_839504), SOCS3 (Cell Signalling, 2923, RRID: AB_2255132), Collagen IV (Bio-Rad, 2150-1470, RRID: AB_2082644), vWF (Thermo Fisher, PA5-16634, RRID: AB_10982615), GFP (Abcam, ab13970, RRID: AB_300798), CD31 (MEC 13·3, BD Biosciences, 550274, RRID: AB_393571), and VEGF receptor 2 (VEGFR2) (clone Avas12, BioLegend, 136406, RRID: AB_2044067). DAPI in an anti-fade mounting medium (Vector Labs, H-1200-10) was used for nuclear staining.

Wang T., Zhou P., Xie X., Tomita Y., Cho S., Tsirukis D., Lam E., Luo H.R, & Sun Y. (2021). Myeloid lineage contributes to pathological choroidal neovascularization formation via SOCS3. EBioMedicine, 73, 103632.

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Multimodal Immunohistochemistry Analysis

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Immunohistochemistry was performed as described previously (Richardson et al., 2012). 10 μm sections were used for all studies. Primary antibodies used: rat ant‐p21 (HUGO291, Abcam ab107099), goat‐anti‐troponin C (Abcam, ab30807), rabbit anti‐p16 (Rockland, 100‐401‐170) and rat anti‐CD31 (MEC13.3, BD Biosciences, 550274). Secondary antibodies used were donkey anti‐rat AF594 (Life Technologies, A21209), donkey anti‐goat AF 488 nm (Life Technologies, A11055), donkey anti‐rabbit AF594 (Life Technologies, R37119), donkey anti‐goat AF488 (Life Technologies, A11055) and donkey anti‐mouse AF647 (A31571, Life Technologies). Slides were mounted in Vectarshield containing DAPI (Sigma, MBD0015). 5‐ethynyl‐2‐deoxyuridine (EdU) labeling performed with Invitrogen Click‐iT EdU Alexa Fluor 594 Imaging Kit (Life Technologies, C10339).

Dookun E., Walaszczyk A., Redgrave R., Palmowski P., Tual‐Chalot S., Suwana A., Chapman J., Jirkovsky E., Donastorg Sosa L., Gill E., Yausep O.E., Santin Y., Mialet‐Perez J., Andrew Owens W., Grieve D., Spyridopoulos I., Taggart M., Arthur H.M., Passos J.F, & Richardson G.D. (2020). Clearance of senescent cells during cardiac ischemia–reperfusion injury improves recovery. Aging Cell, 19(10), e13249.

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Characterization of PECAM Targeting Nanoparticles

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Fluorescein isothiocyanate (FITC)-labeled polystyrene spheres (~125 nm effective diameter) were purchased from Polysciences (Warrington, PA). ¹²⁵I-radiolabled poly(4- vinylphenol) nanoparticle spheres (~145 nm effective diameter) were prepared as previously described.^{55 (link),56 (link)} Purified monoclonal antibodies (mAbs) to human PECAM (huPECAM), clone 62 (mouse IgG2a, referred to as Ab_1h), and clone 37 (mouse IgG1, referred to as Ab_2h), were generously provided by Dr. M. Nakada (Centocor, Malvern, PA).^{26 (link)} The antimouse PECAM (muPECAM) monoclonal antibody clones 390 (rat IgG2a, referred to as Ab_1m)^{57 (link)} and MEC13.3 (rat IgG2a, referred to as Ab_2m)^{58 (link)} were purchased from BD Bioscience (Chicago, IL) and BioLegend (San Diego, CA), respectively. Nontargeted IgG Ab was an isotype mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA). Fluorescent secondary antibody to antihuPECAM were purchased from Invitrogen (Carlsbad, CA). ActinRed 555 reagent was purchased from Life Technologies (Norwalk, CT). Human umbilical vein endothelial cells (HUVECs) endogenously expressing native huPECAM were purchased from Lonza (Walkersville, MD) and maintained in EGM-2 media (Lonza) supplemented with 10% (v/v) fetal bovine serum (FBS).

Chacko A.M., Han J., Greineder C.F., Zern B.J., Mikitsh J.L., Nayak M., Menon D., Johnston I.H., Poncz M., Eckmann D.M., Davies P.F, & Muzykantov V.R. (2015). Collaborative Enhancement of Endothelial Targeting of Nanocarriers by Modulating Platelet-Endothelial Cell Adhesion Molecule-1/CD31 Epitope Engagement. ACS nano, 9(7), 6785-6793.

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Immunohistochemical Analysis of Brain Microvasculature

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Sections were incubated in 0.3 % hydrogen peroxide (30 min), blocked in 10 % rat-serum (2 hr) and incubated with a rat anti-mouse CD31 antibody (MEC13.3, BD Bioscience 1:10, 4 °C overnight). Sections were stained with the Vectastain ABC Kit according to the manufacturer’s protocol (Vector), dehydrated through alcohol series and cleared in xylene with PBS (3X5min) washes in between steps. Slides were digitally scanned and traced (deep layer cortex and subiculum) as for Prussian blue. The Aperio microvessel algorithm (version 11.2.0.780) was optimized to quantify the percent area of tissue and density occupied by blood vessels per brain region on the slide.

Thomas R., Zuchowska P., Morris A.W., Marottoli F.M., Sunny S., Deaton R., Gann P.H, & Tai L.M. (2016). Epidermal growth factor prevents APOE4 and amyloid-beta-induced cognitive and cerebrovascular deficits in female mice. Acta Neuropathologica Communications, 4, 111.

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Tumor Hypoxia and Perfusion Quantification

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Two 10 μm frozen cross-sections from tumor centre were stained for pimonidazole (rabbit anti-pimonidazole antisera, Burlington, USA) and CD31 (rat anti-mouse CD31, clone MEC 13.3, PharMingen/BD Biosciences, Heidelberg, Germany), scanned and blindly analysed as described in detail previously [26 (link)]. After scanning, the same tumor sections were stained with haematoxylin and eosin for identification of viable and necrotic tumour subareas by morphological criteria. The pimonidazole hypoxic fraction (pHF) and the relative vascular area (RVA) were calculated as the percentage of the viable tumor area stained for pimonidazole or CD31, respectively. The fraction of perfused vessels (PF) was calculated as the percentage of the vascular area overlapped with Hoechst 33342 perfusion marker. Necrotic fraction (NF) was determined as the necrotic tumor area divided by the total tumor area. The immunohistochemistry staining protocol for HIF-1α (mouse monoclonal anti-human HIF-1α, BD Biosciences, USA) has previously been described in detail [24 (link), 27 (link)].

Helbig L., Koi L., Brüchner K., Gurtner K., Hess-Stumpp H., Unterschemmann K., Baumann M., Zips D, & Yaromina A. (2014). BAY 87–2243, a novel inhibitor of hypoxia-induced gene activation, improves local tumor control after fractionated irradiation in a schedule-dependent manner in head and neck human xenografts. Radiation Oncology (London, England), 9, 207.

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