In situ detection of O2− and H2O2 were performed as described by Ramel64 (link) with 4-5-week-old seedlings. For in situ detection of O2−, plantlets were immersed and infiltrated under vacuum with 1 mg/ml NBT (N6876, Sigma-Aldrich) staining solution in potassium phosphate buffer (10 mM) with 10 mM NaN3. After infiltration for 2-3 h, stained plantlets were boiled in acetic acid: glycerol: ethanol (1:1:3, v/v/v) solution for 10 min. Samples were then stored in 95% (v/v) ethanol until scanning. O2− was visualized as a blue color produced by NBT reduction to formazan. For in situ detection of H2O2, the staining agent, DAB (D5637, Sigma-Aldrich), was dissolved in H2O and adjusted to pH 3.8 with HCl. The DAB solution was freshly prepared to prevent auto-oxidation. Samples were immersed and infiltrated under vacuum with 1 mg/ml DAB staining solution. Stained plantlets were then boiled in acetic acid: glycerol: ethanol (1:1:3, v/v/v) solution for 10 min, and then stored in 95% (v/v) ethanol until scanning. H2O2 was visualized as a brown color due to DAB polymerization.
D5637
Manufactured by Merck Group
Sourced in United States
The D5637 is a laboratory equipment product manufactured by Merck Group. It is designed to perform specific functions within a laboratory setting. The core function of the D5637 is to facilitate various laboratory processes, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
N6876, by Merck Group (3 mentions)
3 3 diaminobenzidine (dab), by Merck Group (3 mentions)
Lsm 710, by Zeiss (2 mentions)
Envision system hrp labeled polymer, by Agilent Technologies (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Gelatin, by Merck Group (1 mentions)
Anti lamp 1 1d4b, by Abcam (1 mentions)
Goat anti rat alexa 555, by Thermo Fisher Scientific (1 mentions)
Fluoromount g with dapi, by Thermo Fisher Scientific (1 mentions)
Abc kit, by Vector Laboratories (1 mentions)
Ab109497, by Abcam (1 mentions)
Ab64218, by Abcam (1 mentions)
Entellan, by Merck Group (1 mentions)
Image pro plus 5, by Media Cybernetics (1 mentions)
F9887, by Merck Group (1 mentions)
Dm4000b, by Leica (1 mentions)
Ab51253, by Abcam (1 mentions)
Ab7260, by Abcam (1 mentions)
Canada balsam, by Merck Group (1 mentions)
P6418, by Merck Group (1 mentions)
14 protocols using d5637
1
Detecting Reactive Oxygen Species
2
Immunohistochemical Detection of pEtx Antibody
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Endogenous peroxidase activity in the slices was first blocked with 10% methanol (v/v) and 2% H2O2 (v/v) in PBS for 30 min. The slices were pre-incubated for 1 h at RT with PBS containing 20% NGS (Gibco, Paisley, UK), 0.2% Triton, and 0.2% gelatin (Merck, Darmstadt, Germany). Then, the sections were incubated overnight at 4 °C with the rabbit polyclonal anti-pEtx antibody [34 (link)] diluted at 1:100 in PBS containing 1% NGS, 0.2% gelatin, and 0.2% Triton. The samples were washed three times with PBS and incubated with an anti-rabbit EnVision+ system-HRP labeled polymer (#k4001, Dako) for 30 min at RT. The samples were washed three times with PBS, and the sections were developed by a peroxidase reaction, which was performed in a solution containing 0.6 mg/mL of 3,3′-diaminobenzidine substrate (DAB; D-5637, Sigma-Aldrich, Saint Louis, MO, USA) and 0.5 µL/mL of H2O2 in PBS for 5 min, before being stopped with PBS. As a control, sections were treated identically but without incubation with the primary antibody. The sections were counterstained with hematoxylin, and the slides were then dehydrated and mounted with a DPX mounting medium. The slides were examined under a Leica DMD108 digital microimaging system.
3
Visualization of Hydrogen Peroxide in Plants
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The H2O2 staining agent DAB (D5637, Sigma-Aldrich) was dissolved in water, after which the pH was adjusted to 3.8 with KOH. Inflorescences with flowers were treated with 10 μM DAB staining solution under vacuum for 4 hours. Samples were then incubated in 90% ethanol at 70 °C for 10 min to remove chlorophyll. A dark brown color was visualized for H2O2 due to the oxidization of DAB.
4
Immunohistochemical Analysis of Mouse Tissue
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Mouse tissues were embedded immediately after dissection in optimal cutting temperature compound (Tissue-Tek O.C.T.) chilled in a bath of isopentane chilled in a bath of liquid nitrogen. Blocks were stored at −80°C until they were cut at 12 μm and mounted onto slides. Sectioning and PAS-H staining was performed by Histoserv (Germantown, MD, USA). For antibody staining, slides were post-fixed in 4% paraformaldehyde and blocked in eBioscience Blocking Buffer (00-4953-54, Thermo Fisher, Waltham, MA, USA), followed by staining with anti-Lamp1 1D4B (ab25245, Abcam, Cambridge, MA, USA). For fluorescent imaging, slides were then stained with goat anti-Rat Alexa555 (A21434, Thermo Fisher, Waltham, MA, USA), mounted in Fluoromount-G with DAPI (00-4959-52, Thermo Fisher, Waltham, MA, USA), and imaged with a Zeiss LSM 710. For visible light imaging, slides were quenched with 30% hydrogen peroxide prior to blocking as above after the primary antibody, then treated with biotinylated donkey anti-rat IgG (A18743, Thermo Fisher, Waltham, MA, USA) followed by 3,3′-diaminobenzidene (DAB) staining with an ABC kit (PK-6100, Vector Labs, Burlingame, CA, USA) and DAB (D5637, Sigma-Aldrich, St. Louis, MO, USA). Slides were then dehydrated in alcohols, cleared in xylene, coverslipped, and imaged on an Aperio slide scanner. Images were quantified with HALO software.
5
Immunohistochemistry of TSPO in Mouse Brain
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Mouse brains were fixed in 4% PFA, embedded in paraffin, and cut in 5-μm coronal sections. After deparaffinization, brain slices were boiled in citrate buffer (pH = 6, 15 min) and washed three times in deionized water and PBS.
For immunohistochemistry, the sections were incubated in hydrogen peroxide for 10 min to block endogenous peroxidase activity (ab64218, Abcam, Cambridge, UK). Bain sections were incubated with the primary antibody overnight at 4°C: anti-PBR (anti-TSPO) (rabbit, 1:250, ab109497, Abcam, Cambridge, UK). Then, slices were incubated with biotinylated goat anti-rabbit (1:800 in blocking buffer, 45 min, B21078, Life Technologies, Darmstadt, Germany) followed by HRP-streptavidin incubation (1:600 in PBS, 30 min, K1016, DAKO, Hamburg, Germany). The staining was visualized after incubation with 3,3-diaminobenzidine (D-5637, Sigma-Aldrich, St. Louis, USA) for 3 min. Sections were counterstained with hematoxylin, dehydrated, and mounted using Entellan (Merck, Darmstadt, Germany).
For immunohistochemistry, the sections were incubated in hydrogen peroxide for 10 min to block endogenous peroxidase activity (ab64218, Abcam, Cambridge, UK). Bain sections were incubated with the primary antibody overnight at 4°C: anti-PBR (anti-TSPO) (rabbit, 1:250, ab109497, Abcam, Cambridge, UK). Then, slices were incubated with biotinylated goat anti-rabbit (1:800 in blocking buffer, 45 min, B21078, Life Technologies, Darmstadt, Germany) followed by HRP-streptavidin incubation (1:600 in PBS, 30 min, K1016, DAKO, Hamburg, Germany). The staining was visualized after incubation with 3,3-diaminobenzidine (D-5637, Sigma-Aldrich, St. Louis, USA) for 3 min. Sections were counterstained with hematoxylin, dehydrated, and mounted using Entellan (Merck, Darmstadt, Germany).
6
Quantitative Analysis of Apoptosis in Mice Brain
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Mice were anesthetized with isoflurane and then perfused with 50 mL of normal saline through the left ventricle followed by a 50 mL 4% paraformaldehyde solution after decompression. The brain tissue was removed quickly and then post-fixed for 48 hours in 4% paraformaldehyde solution. The brains were serially cut into 25 μm-thick coronal sections from the same region, and then diaminobenzidine (DAB) staining was performed. In brief, sections were incubated at 4°C overnight with rabbit anti-cleaved caspase-3 polyclonal antibody (1:1000; #9662; CST Co., Boston, USA), followed by incubation with a secondary antibody (Goat anti-Rabbit, 1:200, F9887, Sigma-Aldrich Co., St. Louis, Missouri, USA) for one hour; the sections were then incubated with DAB (D5637, Sigma-Aldrich Co., St. Louis, Missouri, USA) solution until brown products appeared under the microscope. Finally, the sections were examined under a microscope (DM 4000B, Leica, Wetzlar, Hesse-Darmstadt, Germany), and then quantitated as the percentage (caspase-3 positive cells / Nissl positive cells) with Image-pro Plus 5.1 software (Media Cybernetics, Bethesda, Maryland, USA).
7
Immunohistochemical Analysis of Phospho-Synuclein in Mouse Brain
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Adult mice were deeply anaesthetized with ketamine:Rompun (3.5:1; 2.5 μl g−1) and perfused with PBS followed by 4% paraformaldehyde (PFA; P6418, Sigma-Aldrich). Excised brains were post-fixed overnight in 4% PFA at 4 °C. Coronal section (40 μm) were cut with a vibratome, rinsed three times in PBS and then once with 3% H2O2 (Sigma-Aldrich) to quench endogenous peroxidase. Sections were washed with PBST (0.1% Triton X-100 in PBS) three times and incubated with blocking solution (4% bovine serum albumin in PBST) for 1 h at room temperature. Sections were incubated overnight in a mixture of rabbit anti-phospho-synuclein (S129) (ab51253, Abcam) and rabbit GFAP (ab7260, Abcam) primary antibodies, diluted 1:1,000 and 1:500, respectively, in blocking solution. The next day, sections were incubated with species-appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h and 30 min at room temperature and then washed three times. Following incubation with an avidin–biotin complex (Vectastain ABC kit; PK6200, Vector Laboratories), immunocomplexes were visualized using 3,3′-diaminobenzidine (DAB; D5637, Sigma-Aldrich) with H2O2. Sections were mounted on gelatin-coated slides using Canada balsam (C1795, Sigma-Aldrich).
8
Immunohistochemical Staining of Microglia in Mouse Brains
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Brain sections (30 μm) from 4% PFA-fixed mouse brains were pre-incubated in 0.3% H2O2 at room temperature (RT) for 30 min, and then in 10% serum at RT for 1 h. Sections were incubated with the primary antibody at 4°C overnight: Iba1 (1:1000, WAKO, 019-19741) or Mac-2 (1:1000, Cedarlane, CL8942AP). After PBS washing, sections were incubated with the secondary antibody: biotinylated goat anti-rabbit IgG (1:400, Vector Laboratories, BA-1000) or biotinylated rabbit anti-rat IgG (1:400, Vector Laboratories, BA-4001) at RT for 1 h, then with avidin-biotin-peroxidase complex (Vector Laboratories, PK-6100) at RT for 30 min, and finally visualized with 3, 3′-diaminobenzidine (DAB, Sigma, D-5637).
9
Visualizing Oxidative Stress in Arabidopsis
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Rosette leaves from 5-week-old Arabidopsis wild-type and acr11 mutant plants were used for trypan blue, DAB, NBT, and SOSG staining as previously described with minor modifications36 (link),37 (link). The DAB (D5637, Sigma-Aldrich) staining solution, 1.25 mg/ml, was freshly prepared in sterilized water and adjusted to pH 3.8 with KOH. Detached rosette leaves were immersed and infiltrated under vacuum with DAB staining solution and then cleared in boiling 95% (v/v) ethanol for 10 min. For NBT staining, detached rosette leaves were immersed and infiltrated under vacuum with 3.5 mg/ml NBT (N6876, Sigma-Aldrich) staining solution in 10 mM potassium phosphate buffer containing 10 mM sodium azide. After vacuum infiltration, stained leaves were bleached in boiling 95% ethanol (v/v) for 10 min. The commercially available fluorescent dye SOSG (S36002, Thermo Fisher) was used to detect singlet oxygen. Rosette leaves from 5-week-old plants were infiltrated with a solution of 100 μM SOSG in 50 mM phosphate potassium buffer (pH 7.5). Plants were exposed to light for 30 min and infiltrated leaves were observed under a 510 META Zeiss confocal laser scanning microscope with excitation at 480 nm and emission at 530 nm.
10
Quantifying Oxidative Stress and Antioxidant Responses
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Various oxidative stress markers [malondialdehyde (MDA); hydrogen peroxide (H2O2) and electrolytic leakage], and antioxidant enzymes [like catalase (CAT); ascorbate peroxidase (APX); glutathione reductase (GR)], proline and RWC (relative water content) were estimated in transgenic lines, VC and WT plants by using the methods described earlier (Garg et al., 2012 (link)). The nitroblue tetrazolium (NBT) (N6876, Sigma-Aldrich) and 3, 3 diaminobenzidine (DAB) (D5637, Sigma-Aldrich) stains were used to check the accumulation of superoxide radicals (O2•–) and H2O2, respectively (Dong et al., 2009 (link)). For O2•– accumulation, seedlings were vacuum infiltrated with 0.1 mg ml-1 NBT in 25 mM Hepes buffer (pH 7.6) and incubated for 2 h at room temperature in the dark. The samples were then transferred to 80% ethanol and treated at 70°C for 10 min. For H2O2 accumulation, seedlings were vacuum infiltrated with 0.1 mg ml-1 DAB in 50 mM Tris-acetate buffer (pH 5.0) and incubated for 24 h at room temperature in the dark before transferring to 80% ethanol (Dong et al., 2009 (link)).
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