Frozen sections were dried for 30 min at 37 °C, post-fixed for 10 min in absolute alcohol then washed in 0.1% TweenTM 20 in PBS (PBST). Endogenous hydrogen peroxidase activity was quenched in 0.3% hydrogen peroxidase (Sigma-Aldrich) and 10% methanol in 0.1% PBST for 30 min. Sections were then washed in 0.1% PBST and blocked in 5% normal goat serum (NGS; Sigma-Aldrich) and 5% BSA in 0.2% PBST for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C in 5% NGS, 5% BSA in 0.2% PBST, then incubated with secondary antibodies for 1 h at room temperature in 5% NGS, 5% BSA in PBST. hydrogen peroxidase activity was detected by avidin/biotin detection method using VECTASTAIN® Elite ABC (Avidin-Biotin) detection system (VECTOR Laboratories, Peterborough, UK). Sections were subjected to 3,3′-diaminobenzidine (DAB) substrate prepared with 0.3% hydrogen peroxidase for appropriate time, before dehydrating through ethanol series to 100% ethanol. Sections were then mounted in Dibutylphthalate Polystyrene Xylene (DPX) mounting solution (MilliporeSigma, St.Louis, MO, USA).
Hydrogen peroxidase
Manufactured by Merck Group
Sourced in United States, United Kingdom
Hydrogen peroxidase is a laboratory reagent used as an oxidizing agent in various analytical and experimental procedures. It is a clear, colorless liquid that decomposes to release oxygen. The core function of hydrogen peroxidase is to serve as a source of active oxygen for chemical reactions and assays.
Automatically generated - may contain errors
Lab products found in correlation
Protein block, by Agilent Technologies (2 mentions)
Antibody diluent, by Agilent Technologies (1 mentions)
Normal goat igg, by Santa Cruz Biotechnology (1 mentions)
Nitrotyrosine, by Merck Group (1 mentions)
Normal goat serum (ngs), by Agilent Technologies (1 mentions)
4 hydroxynonenal, by Abcam (1 mentions)
Clone 2a8, by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Urethane, by Merck Group (1 mentions)
Hexadecyltrimethyl ammonium bromide hetab, by Merck Group (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
Dimethylformamide, by Merck Group (1 mentions)
Avidin biotin block, by Vector Laboratories (1 mentions)
Abc elite, by Vector Laboratories (1 mentions)
Immpact dab, by Vector Laboratories (1 mentions)
Xylene, by Avantor (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Envision plus system hrp, by Agilent Technologies (1 mentions)
7 protocols using hydrogen peroxidase
1
Immunohistochemistry Staining Protocol
2
Endometrial Cancer NFκB Immunohistochemistry
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Immunohistochemical staining of NFκB p65 and p50 were performed on four endometrial cancer sections (two sections were ASE-positive and the other two were ASE-negative) as follows. For pretreatment, sections were blocked by the Protein block (Dako) for 10 min at room temperature and incubated overnight at 4 °C with the primary antibodies, p65 (1:100) and p50 (1:100) (Santa Cruz Biotechnology) diluted in antibody diluent (Dako). Endogenous peroxidase was inactivated using 3 % hydrogen peroxidase (Sigma). The primary antibody in control experiments was replaced with normal goat IgG (Santa Cruz Biotechnology). After incubation with Envision™ + Rabbit/HRP (Dako) for 30 min, signals were developed using 3.3-diaminobenzidine (DAB). The sections were then counterstained with hematoxylin and mounted.
3
Histological Analysis of Liver Necrosis
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Liver tissue was fixed in 10 % formaldehyde (Sigma-Aldrich), dehydrated, embedded in paraffin blocks, and sectioned into 5 μm slices. The sections were stained with hematoxylin and eosin (H&E; Sigma-Aldrich) for histology, and the necrosis grade was evaluated in 20 random 100 × images per animal, as described by Liu et al. [11 (link)] as follows: “0” indicated normal; “1” indicated necrotic cells in the first cell layer adjacent to the central vein; “2” indicated necrotic cells extending two to three cell layers from the central vein; “3” indicated necrotic cells extending three to six layers from the central vein; “4” indicated necrotic cells extending three to six layers and from one central vein to another; and “5” indicated necrotic cells throughout the section. The sections were deparaffinized and dehydrated for immunohistology, and the endogenous peroxide was inactivated with 3 % hydrogen peroxidase (Sigma-Aldrich). The sections were then blocked with 3 % normal goat serum (DAKO, Glostrup, Denmark) for 1 h, stained with primary antibodies against cytochrome P450 subfamily 2E1 (1:200), 4-hydroxynonenal (1:200, Abcam, Cambridge, MA, USA), or nitrotyrosine (1:50, clone 2A8.2, Millipore, Bedford, MA, USA) for 1 h at 37 °C and then incubated with an horseradish peroxidase (HRP)-detection kit (REALTM EnVision, DAKO) according to the manufacturer’s protocol.
4
Anti-Inflammatory Protocol Using EIQ and EIE
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EIQ was kindly supplied by Prof. Whang (Herb Medicine Laboratory, Chung-Ang University, Seoul, Korea) and was dissolved in distilled water for animal treatments. 2,4,6-Trinitrobenzene sulfonic acid (TNBS), sodium carboxymethylcellulose (CMC-Na), 5-aminosalicylic acid (5-AS), EDTA, EGTA, urethane, hexadecyl trimethyl ammonium bromide (HETAB), hydrogen peroxidase, dimethylformamide were purchased from Sigma (St.Louis, MO, USA). EIE was kindly provided by Dong-A Pharmaceutical Co. Ltd (Yong-In, Korea) and was dissolved in CMC-Na for animal treatments. TNF-α and NO assay kits were purchased from Cayman Chemical (Ann Arbor, MI, USA).
5
Paraffin Embedding and Immunohistochemistry
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Formalin-fixed tissues were dehydrated by successive immersion in increasing concentrations (v/v) of ethanol, starting at 70% for 30 minutes; then 75%, 85%, and 95% for 1 hour each; and then 3 times in 100% for 1 hour. Tissues were then cleared with 3 immersions in chloroform for 1 hour, and then infiltrated with 3 immersions in Paraplast tissue-embedding wax.
Paraffin embedded tissue blocks were cut into 4-μm sections using a microtome, and the sections were placed onto Trajan Series 3 microscope slides (Trajan Scientific and Medical, Melbourne, VIC, Australia). Slides were heated for 30 minutes at 80 °C to allow the tissue sections to adhere to the slides. Tissues sections were rehydrated by 2 5 minutes immersions in xylene, followed by 3-minute immersions in 100%, 95%, and 70% ethanol (v/v) consecutively; and a final 1-minute immersion in gentle running tap water. Antigen retrieval was performed by boiling the slides in citrate buffer (pH 6.0) for 15 minutes, then allowing them to cool at RT for 15 minutes, and then rinsing slides in tap water to halt the retrieval process. Endogenous tissue peroxidase activity was quenched by incubating the sections in 0.3% (v/v) hydrogen peroxidase (Sigma-Aldrich) for 5 minutes in a covered container, followed by washing under gently running tap water.
Paraffin embedded tissue blocks were cut into 4-μm sections using a microtome, and the sections were placed onto Trajan Series 3 microscope slides (Trajan Scientific and Medical, Melbourne, VIC, Australia). Slides were heated for 30 minutes at 80 °C to allow the tissue sections to adhere to the slides. Tissues sections were rehydrated by 2 5 minutes immersions in xylene, followed by 3-minute immersions in 100%, 95%, and 70% ethanol (v/v) consecutively; and a final 1-minute immersion in gentle running tap water. Antigen retrieval was performed by boiling the slides in citrate buffer (pH 6.0) for 15 minutes, then allowing them to cool at RT for 15 minutes, and then rinsing slides in tap water to halt the retrieval process. Endogenous tissue peroxidase activity was quenched by incubating the sections in 0.3% (v/v) hydrogen peroxidase (Sigma-Aldrich) for 5 minutes in a covered container, followed by washing under gently running tap water.
6
Immunohistochemistry Protocol for FFPE Sections
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Formal-saline fixed paraffin wax embedded (FFPE) sections were mounted on Snow Coat X-tra charged slides (Surgipath Europe, Peterborough, UK), dewaxed in xylene, rehydrated and subjected to antigen retrieval by pressure cooking for 20 minutes in 10 mM sodium citrate (pH 6.0), before blocking endogenous peroxidase with 3% hydrogen peroxidase (Sigma, Dorset, UK). An avidin–biotin block (Vector Laboratories, Peterborough, UK) and protein block (Dako, Ely, UK) were performed prior to overnight incubation with primary antibodies (Table S2 in File S1 ). Negative controls included incubation with equivalent concentrations of non-specific immunoglobulins and omission of the primary antibody. Sections were then incubated with biotinylated secondary antibody and ABC-Elite (Vector Laboratories). Positive immunolabelling was visualized using 3,3-diaminobenzidine (ImmPACT DAB: Vector Laboratories). Sections were then counterstained counterstained in Mayer's Haematoxylin and mounted with No. 1.5 glass coverslips using Pertex (Cellpath PLC, Hemel Hempstead, UK).
7
Immunohistochemical Analysis of Ovarian Cancer
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Formalin-fixed, paraffin embedded (FFPE) ovarian cancer patient tissue blocks were obtained from the Department of Pathology, Erasmus Medical Center, Rotterdam. The average fixation time was 1–2 y. 4 μm sections were mounted on slides, dewaxed with Xylene (#28979.294, VWR Chemicals) and hydrated. Antigen retrieval was performed in Tris-EDTA buffer (pH 9.0) using a pressure cooker procedure. Slides were incubated in 3% hydrogen peroxidase (#95321, Sigma Aldrich) at RT for 10 min and blocked with 5% milk (#115363, Millipore) in PBS-Tween (#P1379, Sigma Aldrich) for 30 min. Immunohistochemistry was performed using antibodies directed against Tpm1.6/7 (1:100), Tpm1.8/9 (1:100) and mitochondria (1:100, #MAB1273, Sigma Aldrich) followed by the EnVision Plus-HRP system (Dako). Slides were incubated with primary antibodies O/N at 4 °C, washed twice with PBS-Tween and incubated with Rat EnVision+ System-HRP (#P0405, Dako) or Mouse EnVision+ System-HRP (#K4007, Dako) for 30 min. Slides were counterstained with Hematoxylin (#MHS16, Sigma Aldrich).
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