Anti mac3 antibody

Manufactured by BD

Sourced in United States, Japan, United Kingdom

The Anti-Mac3 antibody is a primary antibody that recognizes the Mac3 antigen, which is a membrane-associated glycoprotein expressed on the surface of macrophages and activated monocytes. It is commonly used in immunohistochemical and flow cytometric applications to identify and quantify macrophage populations.

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Lab products found in correlation

Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Anti perilipin antibody, by Abcam (1 mentions) A1r confocal microscope system, by Nikon (1 mentions) Prolong antifade kit, by Thermo Fisher Scientific (1 mentions) Immpact dab peroxidase substrate kit, by Vector Laboratories (1 mentions) Image pro premier software, by Media Cybernetics (1 mentions) Biotinylated secondary antibody, by Agilent Technologies (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Alex fluor 488, by Thermo Fisher Scientific (1 mentions) Anti α smooth muscle actin α sma antibody, by Abcam (1 mentions) Anti neutrophil antibody, by Abcam (1 mentions) Fv1200, by Olympus (1 mentions) Anti cd31, by BioLegend (1 mentions) Fv1200 confocal microscope, by Olympus (1 mentions) Alexa fluor 680, by Thermo Fisher Scientific (1 mentions) Vector red substrate kit, by Vector Laboratories (1 mentions) Anti vcam 1 antibody, by Abcam (1 mentions) Anti icam 1 antibody, by Abcam (1 mentions) Bx40 microscope, by Olympus (1 mentions) 3 amino 9 ethylcarbazole, by Vector Laboratories (1 mentions)

Spelling variants of this product

Anti-Mac3 (18 citations)

10 protocols using anti mac3 antibody

Quantifying Macrophage Accumulation in Adipose Tissue

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For tissue harvest, a 0.9% sodium chloride solution was perfused at a constant pressure via the left ventricle, and then tissues were removed immediately. Epididymal fat pads were fixed with formalin and embedded in paraffin. Tissues were sectioned serially (5 μm) and stained with hematoxylin and eosin for general morphological examination.
For immunohistochemical detection of macrophages, sections were incubated with anti-Mac3 antibody (BD Pharmingen) and stained using the avidin-biotin complex and ImmPACT DAB Peroxidase Substrate Kit (Vector Laboratories). Each section was counterstained with hematoxylin. Macrophage accumulation in fat tissue [Mac3-positive cells (in percentage)] was analyzed using the ratio of the number of Mac3-positive cells to the number of total cells (total number of nucleus) (48 (link)). For perilipin staining, sections were stained with anti-perilipin antibody (Abcam) followed by Alexa Fluor 588–conjugated anti-rabbit immunoglobulin (Ig) antibodies (Molecular Probes). Nuclei were counterstained with Hoechst 33258. The sections were mounted using ProLong Antifade Kit (Molecular Probes) and observed under a confocal microscope (Nikon A1R confocal microscope system; Nikon Instruments Inc.).

Nishimoto S., Fukuda D., Higashikuni Y., Tanaka K., Hirata Y., Murata C., Kim-Kaneyama J.R., Sato F., Bando M., Yagi S., Soeki T., Hayashi T., Imoto I., Sakaue H., Shimabukuro M, & Sata M. (2016). Obesity-induced DNA released from adipocytes stimulates chronic adipose tissue inflammation and insulin resistance. Science Advances, 2(3), e1501332.

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Quantifying Aortic Plaque Composition

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At the time of euthanization, animals were fasted for 4 hours, and blood
was collected from the inferior vena cava. Animals were then perfused with
phosphate-buffered saline (PBS), and tissues were collected. The aortic valve
were embedded in optimal cutting compound (OCT).
Cryosections of embedded aortic roots at the site where all 3 aortic
valve leaflets could be visualized were taken at five-micron intervals.
Sequential sections were stained with Oil-Red O (ORO), picrosirius red (PSR),
anti-Mac3 antibody (BD Pharmingen) or anti-ICAM1 to quantify lipids, necrotic
core, collagen content, activated inflammatory cells or ICAM1 expression in the
aortic root at the level of the aortic valve. Total pixels staining positive for
the component of interest were normalized to overall plaque area to generate
fold increase respect to vehicle-treated mice. Images were collected and
analyzed by a treatment blinded investigator using ImagePro Premier software
(Media Cybernetics). ImagePro Premier software was also used to measure the
necrotic core of each plaque (measured as percentage of the total plaque
area).

Marzolla V., Armani A., Mammi C., Moss M.E., Pagliarini V., Pontecorvo L., Antelmi A., Fabbri A., Rosano G., Jaffe I.Z, & Caprio M. (2017). Essential role of ICAM-1 in aldosterone–induced atherosclerosis. International journal of cardiology, 232, 233-242.

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Lung Macrophage Immunohistochemistry

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Lung-sections were also stained with an anti-Mac-3 antibody (BD Biosciences, San Diego, CA, USA; diluted 1:100), and then were stained with an anti-rat IgG-HRP antibody (Stressgen, CA, USA; diluted 1:200).

Nemoto T., Shibata Y., Inoue S., Igarashi A., Tokairin Y., Yamauchi K., Kimura T., Sato M., Sato K., Nakano H., Abe S., Nishiwaki M., Kobayashi M., Yang S., Minegishi Y., Furuyama K., Machida H, & Kubota I. (2017). MafB silencing in macrophages does not influence the initiation and growth of lung cancer induced by urethane. EXCLI Journal, 16, 914-920.

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Aortic Diameter and Macrophage Quantification

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Aortas were embedded in paraffin and then 5-μm-thick serial sections were prepared for elastic van Gieson (EVG) staining. Digital images of EVG-stained aortas with a reference scale were used for absolute measurement of diameter. Paraffin-embedded aortic sections were also used for immunohistochemical staining. After deparaffinization and blocking, serial sections were incubated with anti-Mac3 antibody (dilution 1:200; BD Pharmingen) for macrophages in mice, followed by biotinylated secondary antibodies (1:200; Dako). For detection, anti-streptavidin-conjugated AlexFluor 488 (1:200; Invitrogen) was used. The nuclei were stained with 4′, 6-diamidino-2-phenylindole (1:5,000; Sigma-Aldrich) after the final series of washes.

Hashizume T., Son B.K., Taniguchi S., Ito K., Noda Y., Endo T., Nanao-Hamai M., Ogawa S, & Akishita M. (2019). Establishment of Novel Murine Model showing Vascular Inflammation-derived Cognitive Dysfunction. Scientific Reports, 9, 4023.

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Wound Healing Histological Analysis

Cited 1 time

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Mice were euthanized via the IP injection of a lethal dose of pentobarbital sodium on days 3, 7, 11, or 14 after wounding. The wound and the surrounding intact skin were harvested, and each sample of wound tissue and the surrounding intact skin was bisected at the center of the wound. The wound samples were stapled onto polypropylene sheets to prevent overcontraction, before being fixed in 4% paraformaldehyde for 18 hours. The samples were then dehydrated in a graded alcohol series, cleaned in xylene, and embedded in paraffin, before 5-μm serial paraffin sections were prepared. At least 6 serial sections from near to the center of the wound were obtained per wound and stained according to the following methods [17 (link)]. Five-μm thick sections were subjected to hematoxylin-eosin (HE) or azan staining or were immunohistologically stained with anti-neutrophil antibody to detect neutrophils (1 : 100; Abcam Japan, Tokyo, Japan), anti-Mac-3 antibody to detect macrophages (1 : 100; BD Pharmingen, Tokyo, Japan), or anti-α-smooth muscle actin (α-SMA) antibody to detect myofibroblasts (1 : 300; Abcam Japan, Tokyo, Japan). Negative control slides were obtained by omitting each primary antibody.

Mukai K., Koike M., Nakamura S., Kawaguchi Y., Katagiri F., Nojiri S., Yamada Y., Miyajima E., Matsumoto M., Komatsu E., Nakajima Y., Urai T., Murakado N, & Nakatani T. (2015). Evaluation of the Effects of a Combination of Japanese Honey and Hydrocolloid Dressing on Cutaneous Wound Healing in Male Mice. Evidence-based Complementary and Alternative Medicine : eCAM, 2015, 910605.

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Quantifying Atherosclerotic Lesions in Mice

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Atherosclerotic lesions were evaluated in a blinded fashion(Bourassa et al., 1996 (link)). Mice were anesthetized with avertin and killed, blood was obtained for analyses and perfusion fixation was performed. The heart and aorta were removed, the heart and proximal aorta were embedded in OCT, serial frozen sections (10 um) of the aortic root were obtained, and 4 to 6 sections per aorta were processed. To evaluate atherosclerotic lesions in en face preparations of the aorta, using a dissecting microscope the adventitial fat was removed and the aorta was opened longitudinally and pinned onto a silicon bed. Lipid staining was performed with Sudan IV(Bourassa et al., 1996 (link)). To evaluate macrophage infiltration in the aortic root, immunohistochemical analysis was done with anti-Mac-3 antibody (BD Pharmingen). Images were captured and areas were determined using Image Pro Plus software(Tangirala et al., 1999 (link); Reddick et al., 1994 (link)).

Umetani M., Ghosh P., Ishikawa T., Umetani J., Ahmed M., Mineo C, & Shaul P.W. (2014). The Cholesterol Metabolite 27-Hydroxycholesterol Promotes Atherosclerosis via Proinflammatory Processes Mediated by Estrogen Receptor Alpha. Cell metabolism, 20(1), 172-182.

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Multimodal Imaging of Tumor Microenvironment

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Frozen blocks of the tumor
in OCT were sectioned
into 14 μm slices on a cryostat. The slices were first imaged
on the Odyssey CLx in the presence of PBS to prevent the tissue from
drying out. This slice was then stained ex vivo with Hoechst 33342
(Thermo Fisher Scientific; Cat. No.H3570), the AF680 agent (due to
its higher affinity), anti-Mac3 antibody (BD Biosciences, San Jose,
CA: Cat. No. 553322) labeled Alexa Fluor555, and anti-CD31 (BioLegends,
San Diego, CA; Cat. No. 102402) labeled Alexa Fluor 488. These slides
were then washed in PBS and imaged on an Olympus FV1200 confocal microscope
equipped with 405, 488, 543, 633, and 748 nm laser lines.
The
integrin image in Figure 4 was post processed to remove stitching artifacts (Figure S2). We used the ImageJ FFT bandpass filter,
suppressing horizontal and vertical lines with a cutoff of 2500 pixels
and 0 pixels with a 5% tolerance.

Bhatnagar S., Verma K.D., Hu Y., Khera E., Priluck A., Smith D.E, & Thurber G.M. (2018). Oral Administration and Detection of a Near-Infrared Molecular Imaging Agent in an Orthotopic Mouse Model for Breast Cancer Screening. Molecular Pharmaceutics, 15(5), 1746-1754.

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Frozen Ankle Tissue Immunofluorescence

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The frozen ankles were sectioned into 10 μm slices on a cryostat using a tungsten carbide blade. The tissue was not fixed and decalcified to avoid loss of fluorescence signal. The slices were stained ex-vivo with an anti-Mac3 antibody (BD Biosciences; San Jose, CA; Cat. No. 553322) labeled with Alexa Fluor 680 (Life Technologies; Carlsbad, CA) and Hoechst 33342 (Thermo Fisher Scientific; Cat. No. H3570). The slides were then washed with PBS and imaged on an Olympus FV1200 confocal microscope with 405, 633 and 750 nm laser lines.

Bhatnagar S., Khera E., Liao J., Eniola V., Hu Y., Smith D.E, & Thurber G.M. (2019). Oral and Subcutaneous Administration of a Near-Infrared Fluorescent Molecular Imaging Agent Detects Inflammation in a Mouse Model of Rheumatoid Arthritis. Scientific Reports, 9, 4661.

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Comprehensive Analysis of Aortic Atherosclerosis

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Atherosclerotic lesions was examined as we reported previously.25 In brief, mice were euthanized with an overdose of pentobarbital and perfused with 0.9% sodium chloride solution. The heart and aorta were removed immediately. The thoracic aorta was opened longitudinally, and the abdominal aorta was snap‐frozen in liquid nitrogen for further analyses. The severity of abdominal aortic aneurysm was determined as reported previously.26 Atherosclerotic lesions in the aortic arch were determined by en face Sudan IV staining. The characteristics of atherosclerotic plaques were examined in frozen sections of lesions in the aortic root (at 5‐μm intervals). Lipid deposition in plaques was determined by oil red O staining. The expression of vascular cell adhesion molecule‐1 (VCAM‐1) and intercellular cell adhesion molecule‐1 (ICAM‐1), and accumulation of macrophages in plaques were examined by immunohistochemical staining. Sections were incubated with anti‐VCAM‐1 antibody (Abcam, Cambridge, MA), anti‐ICAM‐1 antibody (Abcam), or anti‐Mac3 antibody (BD Biosciences, Bedford, MA), followed by the avidin‐biotin complex technique and stained using a Vector Red substrate kit (Vector Laboratories, Burlingame, CA). Each section was counterstained with hematoxylin.

Fukuda D., Nishimoto S., Aini K., Tanaka A., Nishiguchi T., Kim‐Kaneyama J., Lei X., Masuda K., Naruto T., Tanaka K., Higashikuni Y., Hirata Y., Yagi S., Kusunose K., Yamada H., Soeki T., Imoto I., Akasaka T., Shimabukuro M, & Sata M. (2019). Toll‐Like Receptor 9 Plays a Pivotal Role in Angiotensin II–Induced Atherosclerosis. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 8(7), e010860.

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Quantifying Aortic Macrophage Infiltration

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After euthanasia or sudden death, mice were dissected and the aorta (including the brachiocephalic and carotid arteries) was isolated. Arteries were fixed in 4% formaldehyde (pH 7.4) for 24 hours, processed, and embedded in paraffin. Histologic analysis was performed on serial cross sections (5 mm) of the proximal ascending aorta and brachiocephalic and carotid arteries. Macrophages were determined by immunohistochemistry using anti-Mac-3 antibody (Pharmingen, San Diego, CA). Secondary antibody was speciesappropriate horseradish peroxidase conjugates (Vector Laboratories, Burlingame, CA). 3-Amino-9-ethyl-carbazole (Vector Laboratories) was used as a chromogen. Images were acquired with Universal Grab 6.1 (IDL) software (Exelis, Boulder, CO) using an Olympus BX40 microscope (Tokyo, Japan). Images were quantified using ImageJ software (National Institutes of Health, Bethesda, MD).

De Wilde D., Trachet B., Van der Donckt C., Vandeghinste B., Descamps B., Vanhove C., De Meyer G.R, & Segers P. (2015). Vulnerable plaque detection and quantification with gold particle-enhanced computed tomography in atherosclerotic mouse models. Molecular imaging, 14.

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