Monoclonal rat anti cd31 antibody

Manufactured by BD

Sourced in United States

Monoclonal rat anti-CD31 antibody is a laboratory reagent used for the detection and analysis of CD31, also known as platelet endothelial cell adhesion molecule (PECAM-1), which is expressed on the surface of endothelial cells, platelets, and certain immune cells. This antibody can be used in various immunological techniques, such as flow cytometry, immunohistochemistry, and Western blotting, to identify and quantify cells expressing CD31.

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Close spelling variants of this product

Anti-CD31 rat monoclonal antibody (clone MEC13.3) Rat anti-mouse CD31 monoclonal antibody Rat monoclonal anti-CD31 Rat anti-CD31 antibody Rat anti-CD31 Anti-CD31 antibody CD31 antibody Anti-CD31

8 protocols using monoclonal rat anti cd31 antibody

Immunohistochemical Analysis of Kidney Samples

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Immunohistochemistry was performed using frozen sections as described previously [25] (link), [26] (link). The following antibodies were used as primary antibodies: (1) polyclonal rabbit anti-type IV collagen antibody (Chemicon International, Inc., Temecula, CA); (2) polyclonal guinea pig anti-nephrin antibody (Fitzgerald, Concord, MA); (3) polyclonal rabbit anti-ZO-1 antibody (ZYMED Laboratories, Carlsbad, CA) and (4) monoclonal rat anti-CD31 antibody (Pharmingen, San Diego, CA). The glomerular accumulation of monocytes/macrophages was determined by immunohistochemistry using monoclonal rat anti-Mac-2 (lectin, galactoside-binding, soluble, 3) antibody (Cedarlane, Burlington, Ontario, Canada) as previously described [30] (link).
Double immunofluorescent staining was performed as previously described [10] (link), [11] (link). The following antibodies were used as primary antibodies: (1) polyclonal rabbit anti-phosphorylated NF-κB p65 (pNF-κB p65) antibody (Cell Signaling Technology, Danvers, MA); (2) monoclonal rat anti-CD34 antibody (Santa Cruz Biotechnology, CA).

Hinamoto N., Maeshima Y., Yamasaki H., Nasu T., Saito D., Watatani H., Ujike H., Tanabe K., Masuda K., Arata Y., Sugiyama H., Sato Y, & Makino H. (2014). Exacerbation of Diabetic Renal Alterations in Mice Lacking Vasohibin-1. PLoS ONE, 9(9), e107934.

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Multifaceted Immunofluorescence Imaging Approach

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Immunofluorescence was performed using frozen sections (4 μm), as previously described [24 (link)]. The following antibodies were used as primary antibodies: (1) monoclonal rat anti-CD31 antibody (BD Pharmingen, San Diego, CA); (2) polyclonal rabbit anti-type IV collagen antibody (Merck Millipore, Darmstadt, Germany); (3) monoclonal anti-β-galactosidase antibody (Promega, Madison, WI); (4) monoclonal rabbit anti-PDGFRβ antibody (Cell Signaling Technology, Danvers, MA); (5) polyclonal rabbit anti-ZO-1 antibody (Zymed, Carlsbad, CA). Alexa Fluor 488 or 546 (Thermo Fischer Scientific, Waltham, MA) was used as secondary antibodies. In double immunofluorescence, images were obtained using confocal laser microscopy (LSM780; Carl Zeiss, Oberkochen, Germany) in the Central Research Laboratory, Okayama University Medical School.

Masuda K., Tanabe K., Ujike H., Hinamoto N., Miyake H., Tanimura S., Sugiyama H., Sato Y., Maeshima Y, & Wada J. (2018). Deletion of pro-angiogenic factor vasohibin-2 ameliorates glomerular alterations in a mouse diabetic nephropathy model. PLoS ONE, 13(4), e0195779.

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Immunohistochemical Analysis of Brain Vasculature

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For immunohistochemistry, brain sections were washed with 0.1 M phosphate buffer (PB) and then incubated for 30 minutes at room temperature in a blocking solution containing TNB, TBST (0.1%) and normal goat serum (3%). For detection of vascular endothelial cells, sections were incubated overnight at 4 °C with monoclonal rat anti-CD31 antibody (BD Biosciences, 1:50). Tight junction proteins were detected with the following antibodies: mouse anti-Claudin-5 antibody (1:200, ThermoFischer); rat anti-VE-Cadherin antibody (1:100, ThermoFischer) and rabbit anti-ZO-1 antibody (1:100, ThermoFischer). Pericytes were visualized with goat anti-CD13 (1:200; R&D). The primary antibody incubation was followed by 2 h incubation at room temperature with corresponding fluorescence secondary antibodies (1:500, ThermoFisher). DAPI (1:2000 in 0.1 M PB, Sigma) was used to visualize nuclei. Sections were mounted in 0.1 M PB on Superfrost PlusTM microscope slides and coverslipped using Mowiol.

Rust R., Weber R.Z., Grönnert L., Mulders G., Maurer M.A., Hofer A.S., Sartori A.M, & Schwab M.E. (2019). Anti-Nogo-A antibodies prevent vascular leakage and act as pro-angiogenic factors following stroke. Scientific Reports, 9, 20040.

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CD31 Immunohistochemistry on Frozen Sections

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Follow steps from (1) to (6) under Section 3.4 for frozen sections.

Incubate sections with monoclonal rat anti-CD31 antibody (BD Pharmingen, Cat # 550274) diluted at a concentration of 1:100 in blocking buffer for 1 h in a humidified chamber or at 4 °C overnight.

Rinse in PBS, three changes, 5 min each.

Add biotinylated anti-rat secondary antibody diluted to 1:200 in PBS and incubate for 30 min at RT in a humidified chamber.

Rinse in PBS, three changes, 5 min each.

ABC reagent must be prepared 30 min before use for complex formation.

Incubate sections in ABC for 30 min, at RT in humidified chamber.

Rinse in PBS, three changes, 3 min each. Change PBS between washes.

Stain slide with DAB peroxidase substrate (3-3’ diaminobenzidine) for 2-5 min till the desired intensity. Prepare the DAB solution just prior to use.

Menon P, & Fisher E.A. (2015). Immunostaining of macrophages, endothelial cells and smooth muscle cells in the atherosclerotic mouse aorta. Methods in molecular biology (Clifton, N.J.), 1339, 131-148.

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Immunohistochemical Analysis of Neurovascular Markers

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Brain sections were washed with 0.1M phosphate buffer (PB) and incubated with blocking solution containing donkey serum (10%) in PB for 30 min at room temperature. For detection of vascular endothelial cells, sections were incubated overnight at 4°C with monoclonal rat anti-CD31 antibody (BD Biosciences, 1:50). The localization of tight/adherens junction proteins was assessed using the following antibodies: mouse anti-Claudin-5 antibody (1:200, Thermo Fisher Scientific); rat anti-VE-Cadherin antibody (1:100, Thermo Fisher Scientific), and rabbit anti-ZO-1 antibody (1:100, Thermo Fisher Scientific). Pericyte coverage was visualized with goat anti-CD13 (1:200; R&D Systems). The primary antibody incubation was followed by 2 h incubation at room temperature with corresponding fluorescent secondary antibodies (1:500, Thermo Fisher Scientific). Nuclei were counterstained with DAPI (1:2,000 in 0.1 M PB, Sigma). Sections were mounted in 0.1 M PB on Superfrost PlusTM microscope slides and coverslipped using Mowiol.

Weber R.Z., Grönnert L., Mulders G., Maurer M.A., Tackenberg C., Schwab M.E, & Rust R. (2020). Characterization of the Blood Brain Barrier Disruption in the Photothrombotic Stroke Model. Frontiers in Physiology, 11, 586226.

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Immunofluorescence Analysis of Glomerular Markers

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Immunofluorescence was performed using frozen Sections (10 µm). The following antibodies were used as primary antibodies: monoclonal rat anti-CD31 antibody (BD Biosciences); polyclonal rabbit anti-type IV collagen antibody (Abcam); anti-mouse Alk1 antibody (R&D systems); anti-Nephrin antibody (Abcam); anti-WT1 antibody (Abcam); anti-podocin antibody (Abcam); anti-cleaved-caspase 3 (Cell Signaling); anti-PDGFRB (R&D). Alexa Fluor 488 or 647 conjugated antibodies (ThermoFisher Scientific) were used as secondary reagents and slides were mounted with Fluoroshield/DAPI (Sigma). Images were obtained by confocal microscopy (Olympus Fluoview). For quantification of immunofluorescence, staining intensity and area was quantified using 50 randomly selected glomeruli per kidney section. Brightness and contrast were adjusted on displayed images (identically for compared image sets) and quantified (identical threshold settings for compared image sets) using ImageJ. For patient samples, paraffin-embedded tissues were cut into 4- to 6-μm sections and processed for immunofluorescence. Antigen retrieval was performed in citrate solution pH = 6. The sections were then labeled with anti-human Alk1 antibody (R&D systems). Slides were subsequently exposed to specific AF647-conjugated secondary antibody (ThermoFisher).

Lora Gil C., Henley N., Leblond F.A., Akla N., Laurin L.P., Royal V., Gerarduzzi C., Pichette V, & Larrivée B. (2020). Alk1 haploinsufficiency causes glomerular dysfunction and microalbuminuria in diabetic mice. Scientific Reports, 10, 13136.

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Immunohistochemical Analysis of Mmp-9 and CD31

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Paraformaldehyde-fixed paraffin sections were subjected to immunohistochemistry analysis for Mmp-9 using polyclonal goat antieMmp-9 antibody (R&D Systems, Minneapolis, MN). They were followed by reactions with ImmPRESS anti-mouse Ig kit secondary antibody (Vector Laboratories, Burlingame, CA). Immunostaining for CD31 was performed using the multipurpose tissue-processing and paraffinembedding (AMeX) method. 32 Briefly, tissues were fixed in acetone at À20 C overnight, then cleared in methyl benzoate and xylene, consecutively, and embedded in ordinary paraffin at 58 C to 60 C. Paraffin sections were subjected to immunohistochemistry analysis for CD31 with rat anti-CD31 monoclonal antibody (catalog number MEC13.3; BD Biosciences, San Jose, CA), followed by reactions with anti-rat fragment antigen-binding fragments conjugated to horseradish peroxidaseelabeled amino acid polymer (Nichirei Biosciences). Color was developed with diaminobenzidine tetrahydrochloride. After immunohistochemistry analysis, the sections were counterstained with hematoxylin. For assessment of osteoclasts at the chondroosseous junction of the growth plate, numbers of TRAP-or Mmp-9epositive cells were counted at Â400 magnification by an investigator blinded to the samples examined (M.S.).

Shimoda M., Yoshida H., Mizuno S., Hirozane T., Horiuchi K., Yoshino Y., Hara H., Kanai Y., Inoue S., Ishijima M, & Okada Y. (2017). Hyaluronan-Binding Protein Involved in Hyaluronan Depolymerization Controls Endochondral Ossification through Hyaluronan Metabolism. The American journal of pathology, 187(5).

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Immunohistochemical Analysis of YKL-40 and CD31

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Tumors were excised and cryosectioned to 6 μm thickness prior to processing for immunostaining with anti-YKL-40 and anti-CD31 antibodies. In brief, the samples were incubated in 3% H₂O₂ to block endogenous peroxidase activity for 30 min followed by incubation with blocking buffer containing 10% goat serum for 1 h at room temperature. The rAY (1:400) or a rat anti-CD31 monoclonal antibody (1: 500, BD Pharmagen, Mountain View, CA, USA) was incubated at room temperature for 2 h and a goat anti-rabbit or rat secondary antibody (1: 100) conjugated with HRP was then added. Finally, DAB substrate (Dako Inc., Carpinteria, CA, USA) was introduced for several minutes and after washing, methyl green will be used for counterstaining.

Ngernyuang N., Yan W., Schwartz L.M., Oh D., Liu Y.B., Chen H, & Shao R. (2017). A Heparin Binding Motif Rich in Arginine and Lysine is the Functional Domain of YKL-40. Neoplasia (New York, N.Y.), 20(2), 182-192.

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