Liver tissues were fixed in 4% neutral buffered formalin and embedded in paraffin. The paraffin sections were deparaffinized by baking and then dehydrated using xylene and ethanol. After blocking with 1% bovine serum albumin (BSA) for 1 h, the sections were incubated with anti-CD11b (1:100, BD Biosciences, USA), anti-Ly6G (1:100; BD Biosciences), anti-H3cit (1:400; CST, USA), anti-MPO (1:100; Abcam, UK), and anti-N-WASP (1:100; Abcam) antibodies overnight at 4°C. Cells were fixed in 4% paraformaldehyde for 25 min at room temperature and then permeabilized with 0.2% Triton X-100 in PBS for 10 min. The cells were blocked in PBS with 2% BSA for 30 min at room temperature and then incubated with anti-H3Cit (1:400; CST), anti-N-WASP (1:100; Abcam), or anti-MPO (1:100; Abcam) antibodies overnight at 4°C. The sections and cells were then incubated with the indicated Alexa Fluor-conjugated secondary antibodies. DAPI was used to detect nuclei. Between all steps, samples were washed three times in PBS for 5 min each. The sections were visualized using an LSM 800 laser scanning confocal microscope (Zeiss, Germany).
Anti mpo
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-MPO is a laboratory product that detects the myeloperoxidase (MPO) protein. MPO is an enzyme found in the azurophilic granules of neutrophils and monocytes. This product can be used for research purposes to identify and quantify MPO in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd68, by Abcam (5 mentions)
Anti ki67, by Abcam (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Lsm 800 confocal laser scanning microscope, by Zeiss (2 mentions)
Anti ly6g, by Abcam (2 mentions)
Anti cd68 monoclonal antibody, by Bio-Rad (2 mentions)
Anti h3cit, by Abcam (2 mentions)
Anti tnf α, by Abcam (2 mentions)
Dm2500, by Leica (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit hrp, by Agilent Technologies (2 mentions)
Anti n wasp, by Abcam (1 mentions)
Anti ly6g, by BD (1 mentions)
Anti cd11b, by BD (1 mentions)
Primary antibody against tlr4, by Abcam (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Cxcl2, by R&D Systems (1 mentions)
Compound c, by Merck Group (1 mentions)
Anti nrf2, by Santa Cruz Biotechnology (1 mentions)
Sb225002, by Merck Group (1 mentions)
33 protocols using anti mpo
1
Immunohistochemical Analysis of Liver Inflammation
2
Immunohistochemical Analysis of Wound Healing
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Paraffin embedded tissue sections of discarded DFUs, foot skin (FS), oral and skin wounds were used for staining with anti-phospho-STAT3 (1:100; Abcam), anti-MPO (1:1500; Abcam), anti-CD68 (1:800; Abcam), anti-PCNA (1:1000; Cell Signaling), anti-Keratin 5 (1:1000; LSBio), anti-FOXM1 (1:600; Cell Signaling), anti-STAT3 (1:100; Cell Signaling), anti-TNFα (1:25; Abcam), and anti-Ki67 (1:200; Abcam). Murine wounds were excised at day 4 post-wounding and fixed in 4% paraformaldehyde overnight at 4 °C and sections were used for staining with anti-MPO (1:1500; Abcam) and anti-Keratin 5 (1:1000; LSBio). Stainings were visualized with either Alexa Fluor 488-conjugated goat anti-rabbit antibody (1:300; Invitrogen), Alexa Fluor 555-conjugated goat anti-guinea pig antibody (1:300; Invitrogen), Alexa Fluor 647-conjugated goat anti-mouse antibody (1:300; Invitrogen), and mounted with VECTASHIELD antifade mounting media with DAPI (Vectorlabs) to visualize cell nuclei. Specimens were analyzed using a Zeiss LSM 780 confocal microscope and images were acquired with Zen software (Carl Zeiss).
3
Immunofluorescent Staining of Cells and Liver Tissue
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Cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 10 min. After being blocked with 2% bovine serum albumin (BSA) for 30 min at room temperature, the cells were incubated with anti-H3Cit (1:400; CST, USA, 14269S), anti-MPO (1:100; Abcam, UK, ab208670), or anti-F4/80 (1:400; CST, USA, 30325) antibodies overnight at 4 °C. Liver tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The paraffin sections were subsequently prepared and deparaffinized by baking, then dehydrated using xylene and ethanol. After blocking with 1% BSA for 60 minutes, the sections were incubated with anti-H3Cit (1:400; CST, USA), anti-MPO (1:100; Abcam, UK), or anti-F4/80 (1:400; CST, USA) antibodies overnight at 4 °C. The cells and liver sections were then incubated with the indicated Alexa Fluor-conjugated secondary antibodies. DAPI was used to detect nuclei. Between all steps, samples were washed three times with PBS for 5 min each. The sections were visualized using a LSM 800 laser scanning confocal microscope (Zeiss, Germany).
4
Histopathological Analysis of Lung Inflammation
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Lung samples were fixed with 4% paraformaldehyde, embedded in paraffin and cut into 5-μm-thick sections. Hematoxylin and eosin (H&E) staining was used to observe pathological alterations. Inflammatory cell infiltration, including macrophages and neutrophils, was determined with anti-CD68 (Abcam, USA) and anti-MPO (Abcam, USA) immunohistochemical staining, respectively. To show the expression of Toll-like receptor 4 (TLR4), lung sections were incubated with primary antibody against TLR4 (Abcam, USA). Endogenous peroxides were blocked with 3% H2O2, and antigen retrieval was performed by microwave heating post deparaffination and rehydration. Sections were incubated with the primary antibody (1:200) after blocking with 5% bovine serum albumin (BSA) for 30 min. Then, the specimens were stained using a Strept Avidin–Biotin Complex (SABC) kit (Beyotime, China) according to the instructions. Stained sections were photographed using an Olympus inverted microscope (Olympus Imaging America, Center Valley, PA). The percentage of CD68- or MPO-positive cells per field was calculated based on 6–8 random fields.
5
Histological Evaluation of Cardiac Fibrosis
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Hearts were excised, weighted, fixed in 4% paraformaldehyde and embedded in paraffin. 5-μm sections were collected and subjected to sirius red staining to assess collagen deposition. Immunohistochemistry was performed using anti-collagen-Ia1 (Abcam, 1:200), anti-collagen-IIIa1 (Abcam, 1:200), anti-MPO (Abcam, 1:100) antibodies. Then the sections were incubated with biotinylated goat anti-rabbit secondary antibody, detected by DAB.
6
Investigating Nrf2-mediated Anti-inflammatory Mechanisms
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LPS, SB225002, Percoll, and Compound C (CC) were from Sigma-Aldrich (St. Louis, MO). TMZ was from Servier (Tianjin, China). RPMI1640 medium was from Thermo Fisher Scientific (Walham, MA). Nrf2 siRNA was from RiboBio (Guangzhou, China). CXCL2 was from R&D Systems (Minneapolis, MN). DAPI and IL-1β ELISA kit were from Boster (Wuhan, China). Myeloperoxidase (MPO) assay kit and lacate dehydrogenase (LDH) assay kit were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Lipofectamine 2000 was from Invitrogen (Waltham, MA). Protein A/G agroase, anti-Nrf2, and anti-CXCR2 were from Santa Cruz Biotechnology (Dallas, TX). Anti-Ly6G, anti-MPO, anti-GRK2, and isotype antibody were from Abcam (Cambridge, MA). Anti-AMPK and anti-phospho-AMPK were from Cell Signaling Technology (Danvers, MA). Anti-caspase-11 was from Novus Biologicals (Littleton, CO). FITC-CD11b and Percp/Cy5.5-Ly6G were from eBioscience (San Diego, CA). PE-CXCR2 was from BD Biosciences (San Jose, CA). Lysis buffer and BCA protein assay were from Beyotime (Shanghai, China).
7
Protein Radical Detection Using MPO
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The cells were lysed using RIPA buffer containing a protease inhibitor cocktail. The cell lysates were used immediately. Samples were electrophoresed under reducing conditions through 4–12% BisTris NuPage acrylamide gels (Invitrogen, Carlsbad, CA). After electrophoresis, the gels were either stained using Coomassie blue, or transferred to a nitrocellulose membrane and immunoblotted with appropriate antibodies. An Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE) was used for signal detection, which allowed us to simultaneously image MPO with rabbit polyclonal anti-MPO (Abcam, Cambridge, MA) and protein radicals with anti-DMPO antibody.
8
Quantifying Immune Cell Profiles in Tumor, Intestine, and Spleen Tissues
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Immunohistochemical (IHC) staining was performed on tumor, intestinal and spleen tissues to quantify changes in immune cells (CD4 + T, CD8 + T, MPO-positive neutrophils, and F4/80-positive macrophages). The sections were then deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 3 % hydrogen peroxide for 30 min at room temperature. Nonspecific binding was reduced by incubation with 10 % normal goat serum for 30 min. The sections were then incubated with rabbit antibody anti-CD4 (CST, Massachusetts, Cat. 25229, 1:125 dilution), anti-CD8 (Abcam, Cambridge, Cat. ab209775, 1:100 dilution), anti-MPO (Abcam, Cambridge, Cat. ab208670, 1:2000 dilution), or anti-F4/80 (CST, Massachusetts, Cat. 70076; 1:500 dilution) overnight at 4 °C. This was followed by a 45-min incubation with secondary antibodies. Immunoreactivity was revealed using the diaminobenzidine chromogen reaction. The detailed information on primary and secondary antibodies used for IHC staining is summarized in the Supplementary TableA2.
The IHC sections were scanned using a KF-PRO-020 whole slide scanner (KFBIO, Ningbo, China) and then analyzed using the HALO image analysis platform (Indica Labs, Albuquerque, NM, USA), as described in our previous publication [4] (link).
The IHC sections were scanned using a KF-PRO-020 whole slide scanner (KFBIO, Ningbo, China) and then analyzed using the HALO image analysis platform (Indica Labs, Albuquerque, NM, USA), as described in our previous publication [4] (link).
9
Lung Tissue Immunofluorescence Staining
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Lung sections of Sham, CIA, ODE, and CIA+ODE treatment groups previously obtained [11 (link)] were stained with H&E or with anti-CD3 (1:100, Cat#ab5690, Lot#GR3356033-2), anti-CD68 (1:50, Cat#ab31630, Lot#GR3305929-3), and anti-MPO (1:25, Cat#ab9535, Lot#GR331736-4) from Abcam (Cambridge, MA), anti-CD45R (1:40, Cat#14-0452-82, Lot#2178350) from Invitrogen (Grand Island, NY), and anti-CCR2 (1:100, Cat# NBP267700, Lot# HMO537) from Novus Biologicals (Centennial, CO). Cross absorbed (H+L) goat anti-rabbit (Cat#A32731, Lot#UK290266), goat anti-mouse (Cat#A32727, Lot#UL287768) and goat-anti rat (Cat#A21434, Lot#2184321) from Thermo Fisher, Grand Island, NY) were used at 1:100 dilution as secondary antibodies. Slides were mounted with VECTASHIELD® Antifade Mounting Medium with DAPI (Cat#H-1200, Lot#ZG1014, Burlingame, CA) and visualized under Zeiss fluorescent microscope.
10
Liver Histopathology and Biomarker Analysis
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The liver tissues
were stained with hematoxylin and eosin (H&E) and observed with
a light microscope (Zeiss, Germany). Primary mouse antibodies anti-MPO
(1/100; Abcam, Cambridge, UK), the anti-CD68 monoclonal antibody (1:200;
Serotec, Oxford, UK), anti-MMP-9, and anti-VEGF were used to target
neutrophils, Kupffer cells, and liver regeneration-related proteins,
respectively. Anti-TNF-α, anti-Caspase-3, and anti-IL-6 were
also used to identify apoptosis and proinflammatory cytokines.
were stained with hematoxylin and eosin (H&E) and observed with
a light microscope (Zeiss, Germany). Primary mouse antibodies anti-MPO
(1/100; Abcam, Cambridge, UK), the anti-CD68 monoclonal antibody (1:200;
Serotec, Oxford, UK), anti-MMP-9, and anti-VEGF were used to target
neutrophils, Kupffer cells, and liver regeneration-related proteins,
respectively. Anti-TNF-α, anti-Caspase-3, and anti-IL-6 were
also used to identify apoptosis and proinflammatory cytokines.
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