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Peroxidases

Peroxidases are a class of enzymes that catalyze the oxidation of various substrates using hydrogen peroxide as an electron acceptor.
These versatile enzymes play crucial roles in diverse biological processes, including defense against oxidative stress, biosynthesis of various compounds, and signaling pathways.
Peroxidases are found in a wide range of organisms, from microbes to plants and animals, and have numerous applications in various fields such as biochemistry, biotechnology, and medicine.
Research on optimized protocols and products for peroxidases is essential for advancing our understanding and utilization of these important enzymes.
Pubcompare.ai's AI-driven platform can help streamline this research by providing access to relevant protocols from the literature, pre-prints, and patents, as well as enabling intelligent comparisons to identify the best approches.

Most cited protocols related to «Peroxidases»

Immunohistochemical Localization of CYP11B2 and CYP11B1

Cited 44 times

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Publication 2013

Adrenal Glands Alkaline Phosphatase Antibodies Antigens Cells Clone Cells Cloning Vectors Goat Hematoxylin Homo sapiens Immunoglobulins Levamisole Mice, House Paraffin Peroxidases phenylhydrazine Serum Steroid 11-beta-Monooxygenase Technique, Dilution Tromethamine Tween 20

Detecting Apoptosis and Oxidative Stress in Yeast

Cited 42 times

Electron microscopy, annexin V labeling, and DAPI staining were performed as described previously (Madeo et al., 1997 (link)). For the TdT-mediated dUTP nick end labeling (TUNEL) test, cells were prepared as described (Madeo et al., 1997 (link)), and the DNA ends were labeled using the In Situ Cell Death Detection Kit, POD (Boehringer Mannheim). Yeast cells were fixed with 3.7% formaldehyde, digested with lyticase, and applied to a polylysine-coated slide as described for immunofluorescence (Adams and Pringle, 1984 (link)). The slides were rinsed with PBS and incubated with 0.3% H₂O₂ in methanol for 30 min at room temperature to block endogenous peroxidases. The slides were rinsed with PBS, incubated in permeabilization solution (0.1% Triton X-100 and 0.1% sodium citrate) for 2 min on ice, rinsed twice with PBS, incubated with 10 μl TUNEL reaction mixture (terminal deoxynucleotidyl transferase 200 U/ml, FITC-labeled dUTP 10 mM, 25 mM Tris-HCl, 200 mM sodium cacodylate, 5 mM cobalt chloride; Boehringer Mannheim) for 60 min at 37°C, and then rinsed 3× with PBS. For the detection of peroxidase, cells were incubated with 10 μl Converter-POD (anti-FITC antibody, Fab fragment from sheep, conjugated with horseradish peroxidase) for 30 min at 37°C, rinsed 3× with PBS, and then stained with DAB-substrate solution (Boehringer Mannheim) for 10 min at room temperature. A coverslip was mounted with a drop of Kaiser's glycerol gelatin (Merck). As staining intensity varies, only samples from the same slide were compared.
Free intracellular radicals were detected with dihydrorhodamine 123, dichlorodihydrofluorescein diacetate (dichlorofluorescin diacetate), or dihydroethidium (hydroethidine; Sigma Chemical Co.). Dihydrorhodamine 123 was added ad-5 μg per ml of cell culture from a 2.5-mg/ml stock solution in ethanol and cells were viewed without further processing through a rhodamine optical filter after a 2-h incubation. Dichlorodihydrofluorescein diacetate was added ad-10 μg per ml of cell culture from a 2.5 mg/ml stock solution in ethanol and cells were viewed through a fluorescein optical filter after a 2-h incubation. Dihydroethidium was added ad-5 μg per ml of cell culture from a 5 mg/ml aqueous stock solution and cells were viewed through a rhodamine optical filter after a 10-min incubation. For flow cytometric analysis, cells were incubated with dihydrorhodamine 123 for 2 h and analyzed using a FACS^® Calibur (Becton Dickinson) at low flow rate with excitation and emission settings of 488 and 525–550 nm (filter FL1), respectively.
Free spin trap reagents N-tert-butyl-α−phenylnitrone (PBN; Sigma-Aldrich) and 3,3,5,5,-tetramethyl-pyrroline N-oxide (TMPO; Sigma-Aldrich) were added directly to the cell cultures as 10-mg/ml aqueous stock solutions. Viability was determined as the portion of cell growing to visible colonies within 3 d.
To determine frequencies of morphological phenotypes (TUNEL, Annexin V, DAPI, dihydrorhodamine 123), at least 300 cells of three independent experiments were evaluated.

Madeo F., Fröhlich E., Ligr M., Grey M., Sigrist S.J., Wolf D.H, & Fröhlich K.U. (1999). Oxygen Stress: A Regulator of Apoptosis in Yeast. The Journal of Cell Biology, 145(4), 757-767.

Publication 1999

3,3,5,5-tetramethyl-1-pyrroline N-oxide Annexin A5 Antibodies, Anti-Idiotypic Cacodylate Cardiac Arrest Cell Culture Techniques Cell Death Cells cobaltous chloride DAPI deoxyuridine triphosphate dichlorofluorescin dihydroethidium dihydrorhodamine 123 DNA Nucleotidylexotransferase Domestic Sheep Electron Microscopy Ethanol Flow Cytometry Fluorescein Fluorescein-5-isothiocyanate Formaldehyde Free Radicals Gelatins Glycerin Horseradish Peroxidase hydroethidine Immunofluorescence Immunoglobulins, Fab In Situ Nick-End Labeling lyticase Methanol Oxides Peroxidase Peroxidases Peroxide, Hydrogen Phenotype Polylysine Protoplasm pyrroline Rhodamine Sodium Sodium Citrate TERT protein, human Triton X-100 Tromethamine Yeast, Dried

Molecular Modeling of Protein-Ligand Interactions

Cited 26 times

The MM/GBSA or MM/PBSA calculations were applied to six different protein systems, including α-thrombin (7 ligands), avidin (7 ligands), cytochrome C peroxidase (18 ligands), neuraminidase (8 ligands), P450cam (12 ligands) and penicillopepsin (7 ligands). The experimental binding data and the PDB entries for the six proteins are listed in Table S1 in the supporting materials. The chemical structures of the ligands are shown in Figure S1 in the supporting materials. The protonated states for all ligands are shown in Figure 1 in the Supporting Materials.
For ligands bound to α-thrombin, cytochrome C peroxidase, neuraminidase and penicillopepsin, MD simulations were performed based on the crystal structures of the complexes. The starting structures of the six avidin analogues (b2–b7) were generated based on the avidin-biotin complex (PDB entry: 1avd33 (link)). The biotin molecule in the crystal structure was manually mutated to the other ligands. It has been shown that the neutral form of the guanidinium group in b2 and b5 biotin analogues is dominant when it is bound to the protein.34 (link) Therefore, the neutral form of the guanidinium group was used in our simulations. The crystal structures of the nine P450cam ligands were used for MD simulations. Starting structures of the other three P450 ligands (e3, e5 and e6) were obtained by manually modifying the ligand (e1) in the crystal structure of 2cpp35 (link) with the conformation of the protein unaltered. The preparation of the models was accomplished in the SYBYL molecular simulation package.36
In the cytochrome C peroxidase complexes, the lone-pair electrons of the epsilon nitrogen in His175 form resonant bonds with the iron ion and the hydrogen atom is located at the delta nitrogen of His175. In the P450cam complexes, lone-pair electrons of the sulfur atom in Cys357 form resonant bonds with the iron ion and this cysteine residue is thus deprotonated. All the crystal water molecules were kept in the simulations.
The atomic partial charges of all ligands were derived by semiempirical AM1 geometry optimization and subsequent single-point Hartree-Fock (HF)/6-31G* calculations of the electrostatic potential, to which the charges were fitted using the RESP technique.37 The reason why we chose AM1 for optimization, not usually used HF/6-31G(d), is to reduce computational cost.38 (link) The optimization and the electrostatic potential calculations were conducted by Gaussian03.39 Partial charges and force field parameters of the inhibitors were generated automatically using the antechamber program in AMBER9.0.40 (link)
In molecular mechanics (MM) minimizations and MD simulations, the AMBER03 force field was used for proteins41 (link) and the general AMBER force field (gaff) was used for ligands.42 (link) The force field parameters developed by Giammona were used for the heme groups in the cytochrome C peroxidase and the P450cam systems.43 To neutralize the systems, counter ions of Cl− or Na+ were placed in grids that had the largest positive or negative Coulombic potential around the protein. The whole system was immersed in a rectangular box of TIP3P water molecules. The water box was extended 9 Å from solute atoms in all three dimensions.

Hou T., Wang J., Li Y, & Wang W. (2010). Assessing the performance of the MM/PBSA and MM/GBSA methods: I. The accuracy of binding free energy calculations based on molecular dynamics simulations. Journal of chemical information and modeling, 51(1), 69-82.

Publication 2010

Amber Avidin Biotin Camphor 5-Monooxygenase Cysteine Cytochrome c Group Cytochrome c Peroxidase Cytochrome P450 Electrons Electrostatics Guanidine Heme Hydrogen inhibitors Ions Iron Ligands Mechanics Molecular Structure Neuraminidase Nitrogen Peroxidases poly(tetramethylene succinate-co-tetramethylene adipate) Proteins Respiratory Rate Sulfur Thrombin

Adenoviral-Mediated Skin Tissue Analysis

Cited 13 times

Human Samples: Human skin was obtained from corrective plastic surgery. All tissues were obtained according to the guidelines of the University of Pittsburgh and under a protocol approved by the Institutional Review Board of the University of Pittsburgh. Subcutaneous fat tissue was removed and skin tissue was cut into 1.5 cm x 1.5 cm sections. Adenoviral (Ad) constructs were injected intradermally in a volume of 100 µl 1x PBS. Explants containing complete epidermal and dermal layers were cultured in an air liquid interface with the epidermal and keratin layers side up and exposed to air. The culture medium was replaced daily and consisted of Dulbecco’s modified Eagle’s medium (DMEM) (Mediatech, Herndon, VA) supplemented with 10% FBS (Sigma-Aldrich, St Louis, MO), penicillin, streptomycin, and anti-mycotic agent (Invitrogen Life Technologies, Carlsbad, CA). At the indicated time points, skin tissue was harvested and fixed in 10% formalin prior to embedding in paraffin. Skin punch biopsies were obtained from the clinically affected and unaffected skin of patients with SSc as we have previously described [9 (link), 11 (link)].
Adenoviral Constructs: Replication deficient adenoviruses serotype 5 encoding human IGFBP-3, IGFBP-4, or IGFBP-5 were generated as previously described [10 (link)]. Adenovirus serotype 5 lacking cDNA was used as a control. Adenoviruses (1 x 10⁸ pfu) were injected intradermally in a 100 µl volume.
Immunohistochemistry (IHC): Six µm sections of paraffin embedded tissues were deparaffinized and endogenous peroxidases were quenched with 3% H₂O₂. Sections were blocked with 5% serum and incubated with polyclonal anti-IGFBP-5 antibody (Gropep Ltd, Adelaide, Australia) or IgG control antibody (Lab Vision Corporation, Fremont, CA). Sections were washed and incubated with biontinylated secondary antibody (Vector Laboratories, Burlingame CA). Bound secondary antibody was detected using the Vectastain ABC kit (Vector Laboratories) and Zymed AEC Red kit (Zymed, San Francisco CA). A light hematoxylin counterstain was used to identify nuclei using Hematoxylin QS (Vector Laboratories). Images were taken on a Nikon Eclipse 800 microscope (Nikon Instruments Inc., Huntley, IL) using identical camera settings.
Measurement of Skin Dermal and Collagen Bundle Thickness:Six µm sections of paraffin-embedded skin tissue were stained with hematoxylin and eosin (H & E). Images were taken on a Nikon Eclipse 800 microscope. The thickness of the dermis and of individual collagen bundles was measured using Microsuite™ Software (Olympus America Inc.) as we previously described [11 (link)]. Thickness was measured in 5 random fields in each sample. Data are shown in arbitrary units.
Statistical Analysis:Dermal and collagen bundle thickness were analyzed using the Mann-Whitney U test.

Yasuoka H., Larregina A.T., Yamaguchi Y, & Feghali-Bostwick C.A. (2008). Human Skin Culture as an Ex Vivo Model for Assessing the Fibrotic Effects of Insulin-Like Growth Factor Binding Proteins. The Open Rheumatology Journal, 2, 17-22.

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Publication 2008

Adenoviruses Antibodies, Anti-Idiotypic Biopsy Cell Nucleus Cloning Vectors Collagen Cytokeratin Dermis DNA, Complementary DNA Replication Eagle Eosin Epidermis Ethics Committees, Research Formalin Homo sapiens IGFBP3 protein, human IGFBP4 protein, human IGFBP5 protein, human Immunoglobulin G Immunoglobulins Immunohistochemistry Light Microscopy Mycoses Paraffin Embedding Patients Penicillins Peroxidases Peroxide, Hydrogen Plastic Surgical Procedures Serum Skin Streptomycin Subcutaneous Fat Tissues Vision

Microarray Analysis of Permethrin Resistance

Cited 10 times

Only mosquitoes identified as members of A. gambiae s.s. S form and homozygous for the L1014F kdr allele were included in the microarray study. Total RNA was extracted from pools of 10 mosquitoes which were either selected against 0.75% permethrin for the LT₅₀ or not exposed to the insecticide. The quality and quantity of all RNA pools was measured by a spectrophotometer (Nanodrop Technologies) and a random subset was also assessed using a 2100 Bioanalyzer (Agilent Technologies). RNA extraction, amplification and labelling protocols followed those described in Müller et al.[21] (link). Labelled targets were hybridised to an updated version of the A. gambiae detox chip[11] (link),[21] (link) which was printed with a physical rearrangement of the probes (ArrayExpress accession A-MEXP-863). The probes on the microarray include 103 cytochrome P450s, 31 esterases, 35 glutathione S-transferases and 85 additional genes such as peroxidases, reductases, superoxide dismutases, ATP-binding cassette transporters, tissue specific genes and housekeeping genes.
The microarray experiment compared RNA pools from selected vs. unselected mosquitoes, comprising six independent replicates with dye-swaps (12 arrays in total). As each probe was spotted in replicates of four and measurements were obtained for both red and green wavelengths in each array, a total of 96 measurements per probe were obtained. After visual inspection of each array, spot and background intensities were calculated from the scanned array images using GenePix Pro 5.1 software (Axon Instruments). Raw intensities were then analysed with Limma 2.4 software package [27] running in R. Any spot that showed a median intensity in one or both channels at saturation was excluded from the analysis. For each spot background intensities were subtracted (i.e. method = “subtract”) from the total spot intensities and adjusted intensities were transformed into intensity log-ratios and normalised. For the comparison between the two groups, selected vs. unselected, estimates for technical replicates (dye-swaps) were first averaged and then compared between the two groups. A detailed description of the methods used for normalisation and statistical analysis is given in Müller et al.[12] . All microarray data has been deposited in ArrayExpress (accession E-MTAB-52).
In terms of absolute fold change our values are likely to underestimate true fold differences between mosquitoes that would survive an LT₅₀ and those that would not. This is a result of the study design whereby the LT₅₀ survivors were compared with a control group that would be expected to be a mixture of 50% mosquitoes surviving and 50% mosquitoes dying after exposure to 0.75% permethrin. It was not possible to select a fully susceptible control group due to the expected RNA degradation postmortem. The underestimation of fold changes may occur wherever resistant mosquitoes are compared with their parental line. Details of how this study design limits maximum fold change are given in Figure S1. As a consequence we have chosen to rank our genes by statistical significance (i.e., −log₁₀P-value) rather than setting an arbitrary fold change cut-off to filter for candidates.

Müller P., Warr E., Stevenson B.J., Pignatelli P.M., Morgan J.C., Steven A., Yawson A.E., Mitchell S.N., Ranson H., Hemingway J., Paine M.J, & Donnelly M.J. (2008). Field-Caught Permethrin-Resistant Anopheles gambiae Overexpress CYP6P3, a P450 That Metabolises Pyrethroids. PLoS Genetics, 4(11), e1000286.

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Publication 2008

Alleles ATP-Binding Cassette Transporters Autopsy Axon Culicidae Cytochrome P450 detox adjuvant DNA Chips Esterases Genes Genes, Housekeeping Glutathione S-Transferase Homozygote Insecticides Microarray Analysis Oxidoreductase Parent Permethrin Peroxidases Physical Examination RNA Degradation Superoxide Dismutase Survivors Tissue Specificity

Most recents protocols related to «Peroxidases»

Neuroblastoma SOX11 Immunohistochemistry

For NB tissue micro-array, 73 formalin-fixed and paraffin-embedded primary untreated neuroblastoma tumors were used (Supplementary Data 1) from which 5-μm sections were made. Antigen retrieval was done in citrate buffer and endogenous peroxidases were blocked with H₂O₂ (DAKO). The sections were incubated with primary antibodies (1:300 SOX11-C1 antibody, from Prof. Sara Ek, Lund University^{32 (link)} = Antibody 1, 1:50 SOX11 antibody from Klinipath (cat#ILM3823-C01) = Antibody 2), followed by incubation with the Dako REALTM EnvisionTM-HRP Rabbit/Mouse system and substrate development was done with DAB (DAKO). Scanning of the slides was done using the Zeiss Axio Scan.Z1 (Zeiss) and counting of SOX11 positive NB cells was done by H-scoring. In brief, the percentage of SOX11-positive cells is each time multiplied by the intensity (0, 1, 2 of 3) : [1 × (% cells 1 + ) + 2 × (% cells 2 + ) + 3 × (% cells 3 + )]. Blind scoring was done by two independent persons. Each sample was present in triplicate and scores are presented as the average of the three replicates. 15 samples were omitted due to lack of survival data.

Decaesteker B., Louwagie A., Loontiens S., De Vloed F., Bekaert S.L., Roels J., Vanhauwaert S., De Brouwer S., Sanders E., Berezovskaya A., Denecker G., D’haene E., Van Haver S., Van Loocke W., Van Dorpe J., Creytens D., Van Roy N., Pieters T., Van Neste C., Fischer M., Van Vlierberghe P., Roberts S.S., Schulte J., Ek S., Versteeg R., Koster J., van Nes J., Zimmerman M., De Preter K, & Speleman F. (2023). SOX11 regulates SWI/SNF complex components as member of the adrenergic neuroblastoma core regulatory circuitry. Nature Communications, 14, 1267.

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Publication 2023

Antibodies Antigens Buffers Cells Citrates Formalin Immunoglobulins Mus Neoplasms Neuroblastoma Paraffin Peroxidases Peroxide, Hydrogen Rabbits Radionuclide Imaging SOX11 protein, human Tissues Visually Impaired Persons

Immunostaining Analysis of Axonal Transport and Neuronal Integrity

Immunostaining for amyloid precursor protein (APP) was used as a surrogate marker for impairment of axonal transport because it is known to accumulate when transport function in axons is blocked (46 (link)). Neuronal somatodendritic integrity was assessed by immunostaining for the cytoskeletal protein microtubule-associated protein-2 (MAP-2). Assessment of microglia morphology was performed with immunostaining for Iba-1. Immunohistochemistry was performed on 50 µm free-floating sections under moderate shaking. Before staining, the sections were incubated 30 min in 0.3% hydrogen peroxide to quench endogenous peroxidases. After three washing steps in 0.1 M phosphate buffer (pH 7.4), non-specific antibody binding sites were blocked with using 10% normal goat serum. Different free-floating sections were incubated overnight at 4 °C with anti-MAP-2 (1 : 500, monoclonal mouse-IgG; Millipore, MAB3418), anti-APP (1 : 500, monoclonal mouse-IgG; Millipore, MAB348), or anti-Iba-1 (1 : 500, polyclonal rabbit-IgG; Wako, 019-19741) in 5% normal goat serum. After several washes, sections were incubated for 2 h at room temperature with secondary antibodies. For APP and MAP-2 staining, biotinylated anti-mouse-IgG, 1 : 500, Vector, BA9200 was used, and the streptavidin/horseradish peroxidase detection was performed according to the manufacturer's recommendations. These sections were incubated with the substrate diaminobenzidine (DAB, D3939; Sigma-Aldrich Company, St. Louis, MO, United States) for 10 min at room temperature. For Iba-1 staining, Alexa Fluor 594 sary antibody (1:500, Invitrogen, #A-11037) was used. Immunofluorescent images were obtained with a Leica microscope (model DMi8 with THUNDER Imager 3D software).

Wang J., Shi Y., Cao S., Liu X., Martin L.J., Simoni J., Soltys B.J., Hsia C.J, & Koehler R.C. (2023). Polynitroxylated PEGylated hemoglobin protects pig brain neocortical gray and white matter after traumatic brain injury and hemorrhagic shock. Frontiers in Medical Technology, 5, 1074643.

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Publication 2023

Alexa594 Amyloid beta-Protein Precursor anti-IgG Antibodies Axonal Transport Binding Sites, Antibody Buffers Cloning Vectors Cytoskeletal Proteins Fluorescent Antibody Technique Goat Horseradish Peroxidase Immunoglobulins Immunohistochemistry MAP2 protein, human Microglia Microscopy Mus Neurons Peroxidases Peroxide, Hydrogen Phosphates Rabbits Serum Streptavidin Surrogate Markers

Immunohistochemical Analysis of Cartilage Matrix

Immunohistochemistry was performed on deparaffinized and rehydrated sections with primary antibodies directed against type II collagen (mouse antibody #CP18L, Calbiochem, France), antibodies directed against aggrecan (mouse antibody #MA3-16888, ThermoFisher, USA), and NITEGE (mouse antibody #MBS442004, My Biosource, USA) for the detection of aggrecan cleavage. First of all, antigen retrieval was carried out by incubation in either proteinase K (30 min 37 °C, 20 μg/mL #P6556, Sigma Aldrich, USA) or citrate buffer pH6 (10 min 100 °C or 3 h 70 °C) followed by incubation in hyaluronidase (15 min 37 °C, 1 mg/mL, #H3506, Sigma Aldrich, USA), for type II collagen and NITEGE immunostaining, respectively. For aggrecan immunostaining, antigen retrieved was carried out by chondroitinase (30 min at room temperature, 0.25U/mL, #C2905, Sigma Aldrich, USA) after reduction by dithiothreitol (2 h 37 °C, 10 mM, #D9760, Sigma Aldrich, USA) and alkylation by iodoacetamide (1 h 37 °C, 40 mM, #I1149, Sigma Aldrich, USA). Sections were then incubated with 3% (v/v) H₂O₂ (Sigma Aldrich, USA) to inactivate internal peroxidases. After blocking with 2.5% (v/v) horse serum (ref 30 022 #MP-7402 Vector Labs Burlingame, USA) for 30 min, sections were incubated overnight at 4 °C with the primary antibody solution (0.5 μg/mL for type II collagen, 10 μg/mL for anti-aggrecan, and 2 μg/mL for NITEGE in 0.1% (w/v) BSA). The sections were then incubated with peroxidase horse anti-mouse secondary antibodies (ref 30 028 #MP-7402, undiluted, Vector Labs) for 30 min at room temperature. The sections were developed with diaminobenzidine (DAB, #SK-4105, Vector Labs) for 3 min and counterstained using Mayer's hematoxylin (RAL Diagnostic, Martillac, France). Tissue sections were observed using Nanozoomer 2.0 Hamamatsu slide scanner (Hamamatsu Photonics, Hamamatsu, Japan) and imaged with NDP.view2 software® (Hamamatsu Photonics). Type II collagen, aggrecan, and NITEGE immunostaining were semi-quantified using QuPath® software [43 (link)] by measuring the diaminobenzidine (DAB) mean optical density (OD) in the articular cartilage matrix (20 measurements) and using the DAB mean OD in subchondral bone staining, as a blank (3 measurements). Then, the OD values were normalized to the mean intensity of cartilage in CL-sham joints. The results were expressed as an intensity ratio.

Nativel F., Smith A., Boulestreau J., Lépine C., Baron J., Marquis M., Vignes C., Le Guennec Y., Veziers J., Lesoeur J., Loll F., Halgand B., Renard D., Abadie J., Legoff B., Blanchard F., Gauthier O., Vinatier C., Rieux A.D., Guicheux J, & Le Visage C. (2023). Micromolding-based encapsulation of mesenchymal stromal cells in alginate for intraarticular injection in osteoarthritis. Materials Today Bio, 19, 100581.

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Publication 2023

aggrecanase Aggrecans Alkylation Anti-Antibodies Antibodies Antigens Bone Density Buffers Cartilage Cartilages, Articular Citrates Cloning Vectors Collagen Collagen Type II Cytokinesis Diagnosis Dithiothreitol Endopeptidase K Equus caballus Hematoxylin Hyaluronidase Immunoglobulins Immunohistochemistry Iodoacetamide Joints Mus Peroxidase Peroxidases Peroxide, Hydrogen Serum Tissues Type II Mucolipidosis Vision

Immunohistochemical Analysis of Nitrotyrosine

Endogenous peroxidases were quenched (3% hydrogen peroxide in methanol) followed by blocking (4% normal horse serum) for one hour in a humidified chamber. Sections were then incubated with mouse monoclonal anti-nitrotyrosine antibody (1:500, ab61392, Abcam) overnight at 4 °C. The Ultra-Sensitive ABC Peroxidase Mouse IgG Staining Kit (Thermo Fisher Scientific) was used for the application of the secondary antibody as well as the ABC complex. Sections were developed with 3–3′-diaminobenzidine (Sigma-Aldrich) and counterstained with hematoxylin for imaging using light microscopy. Images were captured using a Nikon Eclipse e400 microscope outfitted with a Nikon Coolpix 990 digital camera. Using the 40 × /0.65 N.A. air objective lens, sequential images of the tissue sections were taken to capture the entire length and depth of the articular cartilage across both the medial and lateral femoral condyles/tibial plateaus.

Black A.L., Haskins J., Pozzi A, & Clark A.L. (2023). Sexual dimorphism in reactive oxygen species production and a role for integrin α1β1 in estrogen receptor α and β expression in articular cartilage. Journal of Orthopaedic Surgery and Research, 18, 170.

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Publication 2023

3-nitrotyrosine Antibodies, Anti-Idiotypic Cartilages, Articular Condyle Equus caballus Femur Fingers Hematoxylin Immunoglobulins Lens, Crystalline Light Microscopy Methanol Mice, House Microscopy Peroxidase Peroxidases Peroxides Serum Tibia Tissues Tritium

Visualizing CREB3L2-ATF4 Heterodimers in 5xFAD Mice

CREB3L2-ATF4 heterodimers were visualized using Duolink In Situ Brightfield Detection reagents (DUO92012, MilliporeSigma). CREB3L2 and ATF4 PLA probes were prepared as described for the detection of CREB3L2-ATF4 heterodimers in 5xFAD mice. Per manufacturer’s instructions, the PLA Probe Diluent included in the Probemaker Kit was used in substitution of the PLA Antibody Diluent in the PLA protocol. Before deparaffinization with xylene, slides were placed in a 60°C oven for 1 hour; we proceeded by rehydrating slides using a graded ethanol series (100% > 95% > 70% > 50% > water) plus two 10-min PBS-T washes. Epitope unmasking was done for 20 min in steaming tris-EDTA buffer [10 mM tris base, 1 mM EDTA, and 0.05% Tween 20 (pH 9.0)], followed by three 5-min PBS-T rinses.
We quenched endogenous peroxidases slides with 1% hydrogen peroxide for 30 min before blocking. Costaining of neurofilament (1:400; heavy chain subunit; #N0142, MilliporeSigma) was performed afterward using the Vector Blue Alkaline Phosphatase Substrate Kit (SK-5300, Vector Laboratories). To increase detection sensitivity, we additionally used the Vectastain ABC-AP system (AK-5002, Vector Laboratories) before signal development. Last, sections were dehydrated in a graded ethanol series (50% > 70% > 95% > 100%), cleared with Histo-Clear (64110-01, Electron Microscopy Sciences), mounted in VectaMount (H-5000, Vector Laboratories), and air-dried for 24 hours before proceeding with imaging. Human dorsolateral prefrontal cortex specimens (Brodmann area 8/9; table S2) were manually counted by an experimenter “blind” to the underlying diagnosis. Technical controls: PLA Probe Rabbit IgG Isotype Control MINUS (DUO87004, MilliporeSigma) and CREB3L2 blocking peptide (APrEST73339, Atlas Antibodies). For each case, CREB3L2-ATF4 measurements were interspersed between five randomly selected tissue subregions; specifically, 10 neurons within layers III to V were analyzed in each subregion, for a total of 50 independent measurements per brain.

Gouveia Roque C., Chung K.M., McCurdy E.P., Jagannathan R., Randolph L.K., Herline-Killian K., Baleriola J, & Hengst U. (2023). CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer’s disease gene expression linked to neuropathology. Science Advances, 9(9), eadd2671.

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Publication 2023

Alkaline Phosphatase Antibodies Antigens, CD98 Heavy Chain ATF4 protein, human Brain Brodmann Area 8 Brodmann Area 9 Cardiac Arrest Cloning Vectors Diagnosis Dorsolateral Prefrontal Cortex Edetic Acid Electron Microscopy Epitopes Ethanol Homo sapiens Hypersensitivity III-10 Immunoglobulin Isotypes Immunoglobulins Mice, House Neurofilaments Neurons Peptides Peroxidases Peroxide, Hydrogen Rabbits Tissues Tromethamine Tween 20 Visually Impaired Persons Xylene

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Bx51 microscope by Olympus

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The BX51 microscope is an optical microscope designed for a variety of laboratory applications. It features a modular design and offers various illumination and observation methods to accommodate different sample types and research needs.

Bovine serum albumin (bsa) by Merck Group

Sourced in United States, Germany, United Kingdom, China, Italy, Japan, France, Sao Tome and Principe, Canada, Macao, Spain, Switzerland, Australia, India, Israel, Belgium, Poland, Sweden, Denmark, Ireland, Hungary, Netherlands, Czechia, Brazil, Austria, Singapore, Portugal, Panama, Chile, Senegal, Morocco, Slovenia, New Zealand, Finland, Thailand, Uruguay, Argentina, Saudi Arabia, Romania, Greece, Mexico

Bovine serum albumin (BSA) is a common laboratory reagent derived from bovine blood plasma. It is a protein that serves as a stabilizer and blocking agent in various biochemical and immunological applications. BSA is widely used to maintain the activity and solubility of enzymes, proteins, and other biomolecules in experimental settings.

What are the key applications of Peroxidases?

Peroxidases have a wide range of applications in various fields. In biochemistry, they are used for the detection and quantification of hydrogen peroxide, as well as in the synthesis of various compounds. In biotechnology, peroxidases find use in bioremediation, biosensing, and the production of biofuels. In medicine, they are employed in diagnostic assays and as therapeutic agents for the treatment of oxidative stress-related diseases. Peroxidases are also utilized in the food and cosmetic industries for their antioxidant properties and ability to catalyze the production of desirable compounds.

What are some common challenges in using Peroxidases?

One of the main challenges in using Peroxidases is the need to optimize protocols for specific applications. Factors such as substrate specificity, enzyme activity, and stability can vary significantly depending on the source and type of peroxidase. Additionally, the presence of inhibitors or interfering substances in the sample can affect the performance of peroxidase-based assays. Proper sample preparation and appropriate experimental conditions are crucial for obtaining reliable and reproducible results when working with Peroxidases.

How can PubCompare.ai help in the optimal use and comparison of Peroxidases?

PubCompare.ai's AI-driven platform can be extremely helpful in streamlining research on Peroxidases. The platform allows you to efficiently screen protocol literature from scientific publications, pre-prints, and patents to identify the most relevant and optimized protocols for your specific research needs. The AI-powered analysis can highlight key differences in protocol effectiveness, enzyme characteristics, and experimental conditions, enabling you to choose the best approach for reproducibility and accuracy. This can save you valuable time and resources in your Peroxidase research.

Are there different types or variations of Peroxidases?

Yes, there are several different types and variations of Peroxidases. These enzymes can be classified based on their source (e.g., plant, animal, microbial), structure (e.g., heme-containing, non-heme), or specificity (e.g., lignin peroxidases, manganese peroxidases, chloroperoxidases). Each type of Peroxidase has its own unique properties and applications. For instance, plant Peroxidases are often used in the food and cosmetic industries, while microbial Peroxidases are commonly employed in bioremediation and biosensing. Understanding the differences between Peroxidase types is crucial for selecting the most appropriate enzyme for your research or industrial application.

More about "Peroxidases"

Peroxidases are a vital class of enzymes that play a crucial role in diverse biological processes.
These versatile enzymes catalyze the oxidation of various substrates using hydrogen peroxide as an electron acceptor.
Peroxidases are found in a wide range of organisms, from microbes to plants and animals, and have numerous applications in various fields such as biochemistry, biotechnology, and medicine.
One important application of peroxidases is in immunohistochemistry, where they are commonly used in techniques like the Vectastain Elite ABC kit and Vectastain ABC kit.
These kits utilize biotinylated secondary antibodies and a peroxidase-based detection system to visualize target antigens in tissue samples.
The DAB (3,3'-diaminobenzidine) peroxidase substrate is a popular chromogen used in these techniques, producing a brown staining that can be observed under a microscope like the BX51.
Peroxidases also play a role in the detection of apoptosis, or programmed cell death.
The ApopTag Peroxidase In Situ Apoptosis Detection Kit employs a peroxidase-based system to label and identify apoptotic cells.
Additionally, the DAB Peroxidase Substrate Kit can be used to visualize the activity of peroxidases in various experimental settings.
Overall, the study and optimization of peroxidase protocols and products is essential for advancing our understanding and utilization of these important enzymes.
Researchers can streamline their work by utilizing PubCompare.ai's AI-driven platform, which provides access to relevant protocols from the literature, pre-prints, and patents, as well as enabling intelligent comparisons to identify the best approaches.
By incorporating insights from techniques like the Vectastain kits, DAB, and apoptosis detection kits, researchers can further explore the diverse applications of peroxidases in fields such as biochemistry, biotechnology, and medicine.