Novared peroxidase hrp substrate kit

Manufactured by Vector Laboratories

Sourced in Germany, United States

The NovaRED Peroxidase (HRP) Substrate Kit is a chromogenic substrate for the detection of horseradish peroxidase (HRP) in immunohistochemistry, Western blotting, and other HRP-based applications. The kit provides a soluble, red-colored reaction product upon oxidation by HRP.

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Lab products found in correlation

Mouse anti hif1a 610959, by BD (2 mentions) Power vision poly hrp, by Immunologic (2 mentions) Mouse antihuman c5b 9 tcc, by Quidel (2 mentions) Mouse anti human cd59, by Bio-Rad (2 mentions) Avidin biotin complex, by Vector Laboratories (2 mentions) Eclipse 80i light microscope, by Nikon (1 mentions) Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions) Sequenza racks, by Thermo Fisher Scientific (1 mentions) Digital sight ds 5m, by Nikon (1 mentions) Digital sight control unit, by Nikon (1 mentions) Mayer s haematoxylin, by Merck Group (1 mentions) E cadherin, by Cell Signaling Technology (1 mentions) Ab6640, by Abcam (1 mentions) Vectastain abc hrp kit, by Vector Laboratories (1 mentions) Image pro plus software, by Media Cybernetics (1 mentions) Citrate based antigen unmasking solution, by Vector Laboratories (1 mentions) Anti ucp1 antibody, by Abcam (1 mentions) Normal goat serum (ngs), by Vector Laboratories (1 mentions) Light microscopy, by Zeiss (1 mentions) Anti α syn, by BD (1 mentions)

Spelling variants of this product

NovaRed (137 citations) Peroxidase substrate kit (51 citations) NovaRED substrate (40 citations) NovaRED substrate kit (26 citations) NovaRED peroxidase substrate kit (21 citations) NovaRED peroxidase substrate (15 citations) NovaRed kit (15 citations) NovaRed HRP substrate (5 citations) NovaRED Peroxidase (HRP) Substrate (3 citations) NovaRed HRP substrate kit (2 citations)

10 protocols using novared peroxidase hrp substrate kit

Immunohistochemical Placental Tissue Analysis

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Formalin fixed, paraffin embedded human and mouse placental tissues were sectioned (4 μm), dewaxed and rehydrated through graded alcohols. Haematoxylin and eosin (H&E) staining was performed to identify placental structure. Immunohistochemistry (IHC) was performed as previously described60 (link) with antigen retrieval performed at 99 °C for 20 minutes in EnVision FLEX retrieval solution (high pH, Dako). Slides were incubated in sequenza racks (Thermo Fisher Scientific) overnight at 4 °C with our primary antibodies against TPα and TPβ (1:400; raised in-house)40 (link), E-cadherin (2 μg/mL; Cell Signaling Technology) and PCNA (1 μg/mL; Santa Cruz Biotechnology) or anti-rabbit IgG isotype control at equivalent concentrations. NovaRed peroxidase HRP substrate kit (Vector Laboratories) was used to visualise the staining following the manufacturer’s instructions. Sections were counterstained using Mayer’s haematoxylin (Merck Millipore) and mounted with Eukitt mounting medium (Grale HDS). All placental sections were imaged using a Nikon ECLIPSE 80i light microscope with a Nikon Digital Sight Control Unit and Nikon Digital Sight DS-5M camera at 200x magnification using NIS-Elements software version 3.

Powell K.L., Stevens V., Upton D.H., McCracken S.A., Simpson A.M., Cheng Y., Tasevski V., Morris J.M, & Ashton A.W. (2016). Role for the thromboxane A2 receptor β-isoform in the pathogenesis of intrauterine growth restriction. Scientific Reports, 6, 28811.

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Tumor Tissue Immunohistochemistry for HIF1a

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Frozen tumour sections were stained with either hematoxylin and eosin (H&E) or Alcian blue. Immunohistochemistry was performed on paraffin-embedded tumour sections to stain for HIF1a (mouse anti-HIF1a 610959, BD, 1:100) after antigen retrieval in Tris EDTA/PH9 for 15 minutes at 98 °C. To visualize the immunostaining goat anti-mouse/rabbit/rat Power Vision Poly-HRP (dpvp110HRP, Immunologic) was used as secondary antibody together with the Novared peroxidase HRP substrate kit (sk4800, Vector). As a counterstain Hematoxylin was used.

Lenos K.J., Miedema D.M., Lodestijn S.C., Nijman L.E., van den Bosch T., Romero Ros X., Lourenço F.C., Lecca M.C., van der Heijden M., van Neerven S.M., van Oort A., Leveille N., Adam R.S., de Sousa E Melo F., Otten J., Veerman P., Hypolite G., Koens L., Lyons S.K., Stassi G., Winton D.J., Medema J.P., Morrissey E., Bijlsma M.F, & Vermeulen L. (2018). Stem cell functionality is microenvironmentally defined during tumour expansion and therapy response in colon cancer. Nature cell biology, 20(10), 1193-1202.

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Tumor Tissue Immunohistochemistry for HIF1a

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Macrophage Quantification in Lung Sections

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Macrophage counting was performed in 3-μm sections from paraffin-embedded lungs. After heating at 58°C, lung sections were deparaffinized in xylene and rehydrated. The endogenous peroxidase activity was quenched with 15% (v/v) H₂O₂ in methanol. For staining, a 1:10 dilution of macrophage marker F4/80 antigen-specific antibody (rat monoclonal to F4/80 [ab6640], Abcam, Cambridge, UK) was used. Immune complexes were visualized with a peroxidase-conjugated secondary antibody and NovaRED Peroxidase (HRP) Substrate Kit (Vector labs, LINARIS, Wertheim-Bettingen, Germany) and Hematoxylin solution was used for counterstaining of the sections. The quantification of macrophages and measurement of the lung areas were carried out microscopically using a Qwin macro program from Leica (Wetzlar, Germany). For each lung, the total number of macrophages per lung section was counted. Data were calculated as amount of macrophages/mm².

Seimetz M., Parajuli N., Pichl A., Bednorz M., Ghofrani H.A., Schermuly R.T., Seeger W., Grimminger F, & Weissmann N. (2015). Cigarette Smoke-Induced Emphysema and Pulmonary Hypertension Can Be Prevented by Phosphodiesterase 4 and 5 Inhibition in Mice. PLoS ONE, 10(6), e0129327.

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Immunohistochemical Analysis of TCC and CD59

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The identification and local distribution of TCC and its inhibitor CD59 were assessed by avidin-biotin complex (Vector laboratories) immunohistochemistry (IHC) using NovaRED Peroxidase (HRP) Substrate Kit (Vector laboratories). Antigen retrieval was performed through incubation with hyaluronidase (2 mg/mL in 10 mM citrate buffer, pH 8, 30 min, 37 °C). Sections were incubated with mouse antihuman C5b-9/TCC (1:250, Quidel) or mouse anti-human CD59 (1:125, Bio-Rad) antibodies, overnight at 4 °C, followed by incubation with goat anti-mouse IgG (H + L), Biotin-XX (1:200, Invitrogen) antibody. Isotype controls were also stained with mouse IgG antibody (BioLegend) to prove staining specificity. The stained sections were imaged

Teixeira G.Q., Yong Z., Goncalves R.M., Kuhn A., Riegger J., Brisby H., Barreto Henriksson H., Ruf M., Nerlich A., Mauer U.M., Ignatius A., Brenner R.E, & Neidlinger-Wilke C. (2021). Terminal complement complex formation is associated with intervertebral disc degeneration. European spine journal : official publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society, 30(1).

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Immunohistochemical Analysis of BAFF-R, TACI, and BCMA

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The primary antibodies were rabbit anti-human BAFF-R, goat anti-human TACI, rabbit anti-human BCMA (all from Thermo Fisher Scientific, Waltham, MA, USA), anti-human smooth muscle α-actin (1A4, DAKO, Glostrup, Denmark), and biotinylated anti-human IgD (SouthernBiotech, AL, USA). The secondary antibodies were biotinylated goat anti-IgG (Vector Laboratories, Inc., Burlingame, CA, USA), horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG (H + L) (Thermo Fisher Scientific). Antibody binding was developed using Vectastain ABC HRP Kit, NovaRed Peroxidase (HRP) Substrate Kit, and DAB Peroxidase (HRP) Kit (all from Vector Laboratories).
After microwave heating antigen-retrieval of tissue sections^{50 (link)}, immunohistochemistry assay was conducted as described previously^{3 (link)} and examined by light microscopy (Olympus BX41).
Negative controls with primary antibodies omitted and positive controls (infected tonsil sections and the human B cell line (Raji) were processed in the same manner as above.

Dechkhajorn W., Benjathummarak S., Glaharn S., Chaisri U., Viriyavejakul P, & Maneerat Y. (2020). The activation of BAFF/APRIL system in spleen and lymph nodes of Plasmodium falciparum infected patients. Scientific Reports, 10, 3865.

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Immunohistochemical Analysis of UCP1 in Adipose Tissue

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Staining was done on paraffin-embedded sections of subcutaneous adipose tissue. For antigen retrieval, paraffin-embedded sections were incubated in a citrate-based antigen unmasking solution (Vector Laboratories) for 20 min in a pressure cooker. The sections were blocked in 2.5% normal goat serum (Vector Laboratories) for 30 min followed by a peroxidase block in in 3% H₂O₂ in methanol for 1 h. The sections were incubated in anti-UCP1 antibody (Abcam; ab10983) at 1:250 overnight at 4 °C and were then incubated in secondary antibody, goat anti-rabbit (Vector Laboratories) for 1 h. The sections were then stained using the NovaRED peroxidase (HRP) substrate kit (Vector Laboratories) and counter-stained using hematoxyline solution (Harris modified, Sigma–Aldrich). All photos were taken at a magnification of 20x. The percentage of positive staining was calculated using Image-Pro Plus Software (Media Cybernetics, USA), and an average of four to five photos from two sections were taken per mouse. The average adipocyte size was calculated from 10 adipocytes/slide x 4–5 slides/mouse. All analyses were performed in a blinded fashion.

McCabe K.M., Hsieh J., Thomas D.G., Molusky M.M., Tascau L., Feranil J.B., Qiang L., Ferrante AW J.r, & Tall A.R. (2020). Antisense oligonucleotide treatment produces a type I interferon response that protects against diet-induced obesity. Molecular Metabolism, 34, 146-156.

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Placental Immunohistochemistry for Gal-3 and CK7

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Immunohistochemistry was performed on formalin-fixed, paraffin-embedded placental tissues from both normal and FGR pregnancies as previously described^{19 (link)}. Slides were incubated with gal-3 (0.5 µg/ml; Santa Cruz Biotechnology sc-32790), cytokeratin 7 (0.09 µg/ml; Abcam ab68459) or isotype controls at equivalent concentrations, visualized using NovaRed peroxidase HRP substrate kit (Vector Laboratories) and staining quantitated using ImageJ.

Freitag N., Tirado-Gonzalez I., Barrientos G., Powell K.L., Boehm-Sturm P., Koch S.P., Hecher K., Staff A.C., Arck P.C., Diemert A, & Blois S.M. (2020). Galectin-3 deficiency in pregnancy increases the risk of fetal growth restriction (FGR) via placental insufficiency. Cell Death & Disease, 11(7), 560.

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Immunohistochemical Analysis of TCC and CD59

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TCC deposition and CD59 expression were assessed by avidin-biotin complex (Vector Laboratories) immunohistochemistry using NovaRED Peroxidase (HRP) Substrate Kit (Vector laboratories) as previously described [14] (link). Mouse anti-human C5b-9/TCC (1:250, Quidel) or mouse anti-human CD59 (1:125, Bio-Rad) was used as primary antibodies, followed by incubation with goat anti-mouse IgG (H + L), biotin-XX (1:200, Invitrogen) antibody. Isotype controls were stained with mouse IgG antibody (BioLegend). Sections were imaged with light microscopy (Zeiss). Positive cells for TCC formation (TCC +) and CD59 expression (CD59 +) were counted within a defined region and normalized to the total number of cells within the same region.

Teixeira G.Q., Yong Z., Kuhn A., Riegger J., Goncalves R.M., Ruf M., Mauer U.M., Huber-Lang M., Ignatius A., Brenner R.E, & Neidlinger-Wilke C. (2021). Interleukin-1β and cathepsin D modulate formation of the terminal complement complex in cultured human disc tissue. European spine journal : official publication of the European Spine Society, the European Spinal Deformity Society, and the European Section of the Cervical Spine Research Society, 30(8).

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Bright-field Microscopy Analysis of Protein Localization

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For the bright-field microscopy analysis sections were preincubated first with 1% H₂O₂–10% methanol for 15 min and then with 5% normal goat serum for 1 h. Sections were incubated overnight at room temperature with anti-TH (1:2000; #MAB318, mouse; Millipore) or anti-α-syn (1:1000; #61-787, mouse; BD Laboratories) antibodies. Incubation with biotynilated secondary anti-mouse antibody was followed by incubation with avidin–biotin–peroxidase complex (ABC; Vector Laboratories, Burlingame, CA, USA). Reactions were visualized using NovaRED Peroxidase (HRP) Substrate Kit (Vector Laboratories, Burlingame, CA, USA).

Gully J.C., Sergeyev V.G., Bhootada Y., Mendez-Gomez H., Meyers C.A., Zolotukhin S., Gorbatyuk M.S, & Gorbatyuk O.S. (2016). Up-regulation of activating transcription factor 4 induces severe loss of dopamine nigral neurons in a rat model of Parkinson’s disease. Neuroscience letters, 627, 36-41.

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