Slides (three slides per mouse) loaded with frozen sections were rinsed in PBS, followed by quenching of endogenous peroxidase in 3% hydrogen peroxide for 30 min and permeation by 0.25% Triton™ X-100 in PBS for 20 min. The slides were incubated with blocking reagent (ZSGB-BIO, ZLI-9,022) for 30 min at room temperature. Antigens were then successively detected using the Opal 7-color IHC Kit (NEL797001KT) according to the manufacturer’s instructions. Briefly, slides were incubated with macrophage antibody (MOMA)-2 (Abcam; ab33451, 1:300, Opal 650) and Muscle Actin ( ; CST, 19245, 1:500, Opal 520) for 2 h in a humidified chamber at 37°C, followed by detection using the horseradish peroxidase (HRP)-conjugated secondary antibody (GBI Labs; Polink-1 HRP polymer detection kit) and fluorophore-conjugated TSA® TSA-fluors (PerkinElmer; Opal 7-color IHC Kit, NEL797001KT, 1:100, 20–60 s), after which the primary and secondary antibodies were thoroughly eluted in glycine SDS pH 2 buffer for 40 min at 50°C. Nuclei were subsequently visualized with DAPI (1:2,000), and the slides were coverslipped using ProLong® Gold antifade mountant (ThermoFisher; P36934).
Ab33451
Manufactured by Abcam
Sourced in United Kingdom
Ab33451 is a laboratory equipment product. It is designed for general laboratory use.
Automatically generated - may contain errors
Lab products found in correlation
Ab201340, by Abcam (2 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Spa 895 f, by Enzo Life Sciences (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab5694, by Abcam (2 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Opal 7 color ihc kit, by PerkinElmer (1 mentions)
Histochoice, by Avantor (1 mentions)
Ab11713, by Abcam (1 mentions)
Ab18801, by Abcam (1 mentions)
Af4277, by R&D Systems (1 mentions)
Ab1793, by Abcam (1 mentions)
Ab19134, by Abcam (1 mentions)
Ab9722, by Thermo Fisher Scientific (1 mentions)
Arp58983 p050, by Abcam (1 mentions)
Sc 365858, by Santa Cruz Biotechnology (1 mentions)
Sc 32757, by Abcam (1 mentions)
Ab6671, by Santa Cruz Biotechnology (1 mentions)
Ab86299, by Abcam (1 mentions)
Ab75973, by Abcam (1 mentions)
19 protocols using ab33451
1
Multiplex Immunofluorescence for Macrophage and Smooth Muscle Actin Detection
2
Immunohistochemical Analysis of Aorta and Kidney Tissues
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Aorta and kidney tissues were fixed in Histochoice (VWR, 12 h at 4 °C), rehydrated in 30 % sucrose/1XPBS (12 h at 4 °C), embedded in TFM (Fisher Scientific), and sectioned on the cryostat at 7 μm. Sections were prepared for immunofluorescent detection of either AT1 receptor (1:1000 dilution; #ab18801, Abcam, Cambridge, MA), AT2 receptor (1:1000 dilution; #ab19134, Abcam), MOMA-2 (1:1000 dilution; #ab33451, Abcam), renin (1:500 dilution; # AF4277, R&D Systems), or TNF-α (1:1000 dilution; #ab1793, Abcam) and/or von Willebrand factor (vWF -aorta sections only – 1:1000 dilution; #ab11713, Abcam), using anti-sheep Alexa Fluor 488 or anti-goat Alexa Fluor 647 and secondary antibodies (double the concentration of primary antibody used; Thermo Fisher Scientific). All slides were imaged and analyzed using Image J software (NIH, Bethesda, MD), as previously described by our laboratory [45 (link)]. Total fluorescence was quantified per unit area in kidneys and aortas; colocalization (endothelial cells of the aorta) was determined by quantifying the overlaid signals’ fluorescence from a minimum of 6 sections on 3 slides (n = 3–5 per group).
3
Macrophage Apoptosis in Carotid Plaque
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Human carotid plaque and murine aortic root sections were double stained for macrophages and apoptotic cells. Macrophages were labeled with anti-CD68 Ab (Abcam ab201340 1:100) or anti-MOMA-2 Ab (Abcam ab33451 1:50) antibody and apoptotic cells were stained with TUNEL kits (Progema G3250) according to the manufacturer’s instructions. The proportion of TUNEL+ in CD68+ cells in each sample was calculated as the apoptosis rate of macrophages in the plaque. Nine randomized magnified fields were chosen to measure the apoptosis rate.
4
Plaque Characterization and Vulnerability Index
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The paraffin sections were incubated with primary antibodies against mouse anti-rabbit NLRP3 (Aviva Systems, ARP63297_P050), caspase1 (Aviva Systems, ARP58983_P050), IL- 1β (abcam, ab9722), IL-18(Invitrogen, PA5-79479), MOMA-2 (abcam, ab33451) and α-SMA (CST19245) monoclonal antibody response. The results were analyzed by using a computer-aided morphometric analysis system (Image-Pro Plus 6.0, Media Cybernetics, United States). Measure at least three slices of each sample and take the average. The vulnerability index was calculated as (macrophage staining% + lipid staining%)/(SMC% + collagen fiber%) (Shiomi et al., 2001 (link)). Plaque rupture is defined as a plaque with deep injury with a real defect or gap in the fibrous cap that had separated its lipid-rich atheromatous core from the flowing blood, thereby exposing the thrombogenic core of the plaque (Schaar et al., 2004 (link)).
5
Murine Carotid Artery Histological Analysis
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The murine carotid artery tissues obtained were fixed with 4% paraformaldehyde overnight. The samples were then embedded with OCT compound (Sakura Tissue Tek), frozen, and sectioned at a thickness of 6 μm per section. For histological analysis, the sections were stained with hematoxylin and eosin and observed by light microscopy (Olympus BH‐2). Immunofluorescent staining was performed with rat anti‐MOMA‐2 (ab33451; Abcam) and donkey anti–rat Alexa 488 (Molecular Probes, Invitrogen). TO‐PRO‐3 stain (Molecular Probes, Invitrogen) was used to identify nuclei. Fluorescent staining was visualized, and digital images were taken on a Nikon A1R Laser Scanning Confocal imaging system with the appropriate argon‐beam lasers.
6
Quantifying Inflammatory Proteins in Tissues
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Immunofluorescent staining was used to quantify TNF-α, IL-10, nitrotyrosine, MOMA-2, and NF-κB p65 proteins using techniques previously described by our laboratory [86 (link)]. Tissues were stained using the following primary antibodies: TNF-α (1:250; Abcam, ab6671), IL-10 (1:200; Santa Cruz Biotechnology SC-365858), nitrotyrosine (1:200; Santa Cruz Biotechnology, SC-32757) MOMA-2 (1:500, Abcam ab33451), and NF-κB p65 (1:500, Abcam, ab86299). Secondary antibodies used were anti-rabbit Alexa Fluor 555, anti-mouse Alexa Fluor 546, anti-mouse Alexa Fluor 555, anti-rabbit Alexa Fluor Plus 488, and anti-rat Alexa Fluor 488. Slides were imaged under fluorescent microscopy at 40x with the appropriate excitation/emission filter, digitally recorded, RGB overlay signals were split and analyzed for specific fluorescence using image densitometry with Image J software (NIH). A minimum of 3–4 locations on each section (4 sections per slide), 3 slides, and n = 3 per group were used for analysis; 40x images were used for image quantification.
7
Immunohistochemical Analysis of Macrophage Markers
8
Immunohistochemical Analysis of Aortic Macrophages
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Frozen aortic sinus sections were dried and fixed in cold acetone for 10 min and were repaired using sodium citrate–EDTA. Aortic sinus sections were then washed with PBS buffer (pH = 7.4) three times and blocked with 2% BSA for 1 h at 37 °C. Then, the sections were incubated with rabbit anti-mouse MOMA (Monocyte & Macrophage) (1:50, ab33451, Abcam, Shanghai, China) primary antibodies at 4 °C overnight and with Alexa Fluor® 488-conjugated goat anti-rabbit (1:200, ab150077, Abcam) secondary antibodies for 50 min at 25 °C. After being washed with PBS buffer three times, the sections were incubated with DAPI (62248, Thermo Scientific). After the sections were washed and slightly dried, they were mounted with an anti-fluorescence quenching mounting medium. A Nikon upright fluorescence microscope (Nikon) was used to observe and obtain fluorescence images. The positive areas of the sections were quantified using Image-Pro Plus (v 6.0) software.
9
Quantifying Plaque Composition in Atherosclerosis
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Smooth muscle cell (SMC) and macrophage content were assessed within the entire plaque area, as previously described (Lemaire et al. 2011 (link)). Briefly, slides were rinsed with PBS and blocked with 3% bovine serum albumin for 1 h. Next, they were incubated with primary antibody for 1 h at room temperature; 1:100 for monoclonal muscle cell actin [clone 1A4] (Abcam; ab7817), 1:50 for Moma-2 (Abcam; ab33451), rinsed, and incubated with fluorescently labeled secondary antibodies at room temperature for 1 h. A 1:500 dilution each of goat antimouse (Alexa 488; A11001) and a goat antirat (Alexa 488; A11006) (Invitrogen) was used to visualize muscle cell actin and macrophages respectively. Images were acquired using Infinity Capture software and a Lumenera camera. The presence of the immunofluorescent marker from three to five sections per animal was quantified using ImageJ software (Schneider et al. 2012 (link)) and expressed as a percentage of the total lesion area to define the contribution of each component to plaque independent of plaque size.
10
Immunohistochemical Staining of Frozen Liver
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