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Novared substrate

Manufactured by Vector Laboratories
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NovaRED substrate is a chromogenic detection system used for immunohistochemistry and immunocytochemistry applications. It produces a bright red color precipitate upon reaction with the target antigen.

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Lab products found in correlation

Citric acid buffer, by Vector Laboratories (3 mentions) Abc elite kit, by Vector Laboratories (3 mentions) Vector elite abc hp kit, by Vector Laboratories (2 mentions) Axio observer z1 inverted microscope, by Zeiss (2 mentions) Orca er ccd camera, by Hamamatsu Photonics (2 mentions) Axiovision software, by Zeiss (2 mentions) Impress anti rabbit hrp, by Vector Laboratories (2 mentions) Sc 9168, by Santa Cruz Biotechnology (2 mentions) Immunospot plate reader, by Cellular Technology (2 mentions) Axiovert, by Zeiss (2 mentions) Insulin, by Abcam (2 mentions) Somatostatin, by Agilent Technologies (2 mentions) Pancreatic polypeptide, by Agilent Technologies (2 mentions) Glucagon, by Agilent Technologies (2 mentions) Tnf α, by Abcam (2 mentions) Proinsulin, by Progen Biotechnik (2 mentions) Vectastain elite abc kit, by Vector Laboratories (2 mentions) Vectastain abc hrp kit, by Vector Laboratories (2 mentions) Anti myc tag, by Miltenyi Biotec (2 mentions) Antigen unmasking solution, by Vector Laboratories (2 mentions)

Spelling variants of this product

NovaRed (137 citations) VECTOR NovaRED Peroxidase (HRP) Substrate Kit (17 citations) NovaRED peroxidase substrate (15 citations) Vector NovaRED peroxidase substrate (13 citations) Vector NovaRed substrate (11 citations) ImmPACT NovaRED Peroxidase (HRP) Substrate (8 citations) ImmPACT NovaRED Peroxidase Substrate Kit (7 citations) NovaRED Substrate KIT FOR PEROXIDASE (3 citations) NovaRed HRP substrate kit (2 citations) NovaRED substrate of an immunohistochemistry kit (2 citations)

40 protocols using novared substrate

1

Tissue Microarray Immunohistochemistry for ATP1A1

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Paraffin-embedded tissue microarray (TMA) slides, BR1201, were purchased from US Biomax (Rockville, MD, USA). The slides were de-paraffinized, followed by antigen retrieval in sodium citrate buffer, pH 6.8 (ScyTek Laboratories, Logan, UT, USA), for 20 min in a microwave oven. TMA specimens were probed with an anti-ATP1A1 antibody (Abcam, USA) using the VECTASTAIN ABC kit (Vector Labs, Newmark, CA, USA), with staining visualized with a NovaRED substrate (Vector Labs), followed by counterstaining with hematoxylin. The stained slides were subsequently scanned using a ZEISS Axio Scan Z1 microscope (Carl Zeiss Microscopy, Oberkochen, Germany), with staining intensity calculated by the IHC profiler [27 (link)] plug-in in Image J software.
Kim Y., Ko J.Y., Kong H.K., Lee M., Chung W., Lim S., Son D., Oh S., Park J.W., Kim D.Y., Lee M., Han W., Park W.Y., Yoo K.H, & Park J.H. (2024). Hypomethylation of ATP1A1 Is Associated with Poor Prognosis and Cancer Progression in Triple-Negative Breast Cancer. Cancers, 16(9), 1666.
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2

Visualization of Versican and Hyaluronan

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IHC for versican and affinity histochemistry for hyaluronan were performed with paraffin-embedded tissue. After deparaffinization, endogenous peroxidases were blocked using H2O2 in methanol and tissue sections were rehydrated in a series of graded ethanol. For versican IHC, sections were digested with 0.2 U/ml of chondroitinase ABC to remove chondroitin sulfate side chains and expose versican epitopes. Tissues were then incubated with 5% Carnation non-fat milk in PBS for tissue blocking, followed by overnight incubation with either rabbit anti-versican polyclonal antibody or isotype-matched control IgG. This was followed by biotinylated goat anti-rabbit IgG antibody for 2 h at room temperature. The tissue sections were rinsed twice with PBS and then incubated with the Vector “Elite” ABC-HP kit (Burlingame, CA) in a moist chamber for 30 min at room temperature. Detection was performed using the Vector NovaRed substrate (Burlingame, CA) for 10 min at room temperature. Slides were counterstained with Gills #3 hematoxylin. The same protocol was used for hyaluronan affinity histochemistry, except that the tissue was not treated with chondroitinase ABC and biotinylated hyaluronan binding protein (b-HABP, the N-terminal hyaluronan binding region of aggrecan which has been biotinylated) was used instead of the primary and secondary antibodies.
Chang M.Y., Tanino Y., Vidova V., Kinsella M.G., Chan C.K., Johnson P.Y., Wight T.N, & Frevert C.W. (2014). A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease. Matrix biology : journal of the International Society for Matrix Biology, 35, 162-173.
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3

Quantifying Pancreatic Beta Cell Area

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Pancreata were isolated from control and STZ-treated dams after delivery of pups, and from neonatal male and female rats on postnatal day 1. Specimens were fixed in formalin overnight, embedded in paraffin and sectioned by the Histology Core of the Department of Anatomy and Cell Biology at the Indiana University School of Medicine. Staining for insulin was performed (sc-9168, 1:500, Santa Cruz Biotechnology, Dallas, TX), with detection using Impress anti-rabbit HRP and Novared substrate (Vector Laboratories, Burlingame, CA). A Zeiss Axio Observer Z1 inverted microscope (Zeiss, Thornwood, NY) equipped with an Orca ER CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan) was used to acquire digital images of the entire stained longitudinal pancreatic section. The beta cell area of at least 5 sections, each separated by at least 75 μm, from at least 3 animals in each group was calculated using Axio-Vision Software (Zeiss, Thornwood, NY) as previously described (48 (link)).
Blue E.K., Ballman K., Boyle F., Oh E., Kono T., Quinney S.K., Thurmond D.C., Evans-Molina C, & Haneline L.S. (2014). Fetal hyperglycemia and a high fat diet contribute to aberrant glucose tolerance and hematopoiesis in adulthood. Pediatric research, 77(2), 316-325.
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4

Quantifying Pancreatic Beta Cell Area

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Pancreata were isolated from control and STZ-treated dams after delivery of pups, and from neonatal male and female rats on postnatal day 1. Specimens were fixed in formalin overnight, embedded in paraffin and sectioned by the Histology Core of the Department of Anatomy and Cell Biology at the Indiana University School of Medicine. Staining for insulin was performed (sc-9168, 1:500, Santa Cruz Biotechnology, Dallas, TX), with detection using Impress anti-rabbit HRP and Novared substrate (Vector Laboratories, Burlingame, CA). A Zeiss Axio Observer Z1 inverted microscope (Zeiss, Thornwood, NY) equipped with an Orca ER CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan) was used to acquire digital images of the entire stained longitudinal pancreatic section. The beta cell area of at least 5 sections, each separated by at least 75 μm, from at least 3 animals in each group was calculated using Axio-Vision Software (Zeiss, Thornwood, NY) as previously described (48 (link)).
Blue E.K., Ballman K., Boyle F., Oh E., Kono T., Quinney S.K., Thurmond D.C., Evans-Molina C, & Haneline L.S. (2014). Fetal hyperglycemia and a high fat diet contribute to aberrant glucose tolerance and hematopoiesis in adulthood. Pediatric research, 77(2), 316-325.
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5

Ki67 Immunohistochemistry in Paraffin Kidney Sections

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Paraffin-embedded kidney sections were dewaxed in xylene and rehydrated in a graded series of ethanol. Slides were incubated in 0.92% citric acid buffer (Vector Laboratories, Burlingame, CA) at 95°C for 15 min. After cooling to room temperature, the slides were incubated with 2% H2O2 in 60% methanol and blocked in 2% normal rabbit serum/TBS, after which they were incubated with goat anti-Ki67 antibody (1:50, Santa Cruz Biotechnologies) in 1% normal rabbit serum/TBS with 0.02% Triton X-100 at 4°C overnight. The detection was carried out as per the instructions provided by an immunohistochemistry kit with NovaRED substrate (Vector Laboratories).
Kuncewitch M., Yang W.L., Corbo L., Khader A., Nicastro J., Coppa G.F, & Wang P. (2015). WNT AGONIST DECREASES TISSUE DAMAGE AND IMPROVES RENAL FUNCTION AFTER ISCHEMIA-REPERFUSION. Shock (Augusta, Ga.), 43(3), 268-275.
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6

IFN-γ ELISpot Assay for Immune Functionality

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Cryopreserved cells were thawed, counted, and resus-pended in complete medium (R10) RPMI 1640 (Gibco; Grand Island, NY) supplemented with 10% fetal bovine serum (Gemini Bio-Products; West Sacramento, CA) and 5% Pen-Strep-Glut (Gibco; Grand Island, NY). Cell suspensions at 2 × 106 cells/mL were incubated overnight at 37 °C, 5% CO2 in 50 mL tubes. The IFN-γ ELISpot assay was performed using Mabtech pre-coated plates (Mabtech; Mariemont, Ohio); Mabtech biotinylated anti-IFN-γ 7-B6-1 monoclonal antibody (Ab) was used as the detecting Ab; NovaRED substrate (Vector Laboratories; Burlingame, CA) was used to reveal the presence of spots. Spots formed by IFN-γ-secreting cells were counted with an automated ImmunoSpot plate reader (Cellular Technologies, Cleveland Ohio), and results are presented as spot-forming cells (SFC) as indicated in each figure. For all samples, the cells were evaluated for their functionality using the CEF and CMVpp65 peptide pools at the concentration of 1 µg/mL. For the HIV-1 seropositive samples, additional testing was performed using the PTE peptides. This assay was performed following Good Clinical Laboratory Procedures guidelines; detailed Standard Operating Procedures are available upon request.
Sambor A., Garcia A., Berrong M., Pickeral J., Brown S., Rountree W., Sanchez A., Pollara J., Frahm N., Keinonen S., Kijak G.H., Roederer M., Levine G., D’Souza M.P., Jaimes M., Koup R., Denny T., Cox J, & Ferrari G. (2014). Establishment and maintenance of a PBMC repository for functional cellular studies in support of clinical vaccine trials. Journal of immunological methods, 0, 107-116.
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7

Immunostaining for Phospho-SMAD2/3

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Autoclaved 22x22 mm cover glasses (3506; Thermo Fisher Scientific) were placed into wells prior to cell seeding and cytokine treatments that were previously described. Following treatment, cells on cover glasses were fixed in 4% paraformaldehyde for 15 minutes and thoroughly washed with DPBS. Cells were permeabilized with ice-cold 100% methanol for 10 minutes at −20°C. Cells were blocked for endogenous peroxidase activity with Bloxall (SP-6000; Vector Labs) for 10 minutes. Cells were then blocked with 5% goat serum (Sigma) for 1 hour and incubated in anti-Phospho-SMAD2/3 (1:100; D27F4; Cell Signaling) primary antibody overnight at 4°C. Cells were then incubated in biotinylated goat-anti-rabbit secondary (1:500; B2770; Invitrogen) for 45 minutes at 37°C and subsequently exposed to ready-to-use peroxidase (PK-7100; Vector Labs). Color was developed using Nova Red substrate (SK-4800; Vector Labs). Micrographs were captured with a Nikon Labophot-2 microscope with Nuance software version 3.0.0.
Tran L.L., Dang T., Thomas R, & Rowley D.R. (2021). ELF3 mediates IL-1α induced differentiation of mesenchymal stem cells to inflammatory iCAFs. Stem cells (Dayton, Ohio), 39(12), 1766-1777.
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8

Immunohistochemical Analysis of Postnatal Tendon Maturation

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For immunohistochemisty, mouse hindlimbs were collected during postnatal tendon maturation from P10 to P60 and fixed in 10% neutral buffered formalin overnight at 4 °C. Samples were paraffin-embedded and sectioned at 6 μm, following decalcification in 10% (w/v) EDTA (pH 7.4) for 2 weeks with daily solution changes. The paraffin sections of the patellar and Achilles tendon incubated with antibodies against Rcn3 (Abcam, ab204178) at 4 °C overnight. The sections were then incubated in anti-rabbit-biotin antibody (Jackson Immuno, 711-065-152) for 30 min at RT, rinsed with PBS, and incubated with streptavidin-HRP (Jackson Immuno, 016-030-084) for 30 min at RT. Bounded antibodies were detected using NovaRed substrate (Vector Laboratories, SK-4800).
Park N.R., Shetye S.S., Bogush I., Keene D.R., Tufa S., Hudson D.M., Archer M., Qin L., Soslowsky L.J., Dyment N.A, & Joeng K.S. (2021). Reticulocalbin 3 is involved in postnatal tendon development by regulating collagen fibrillogenesis and cellular maturation. Scientific Reports, 11, 10868.
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9

Histological Analysis of Tuberculosis in Mice

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Animal experiments were performed in accordance with the Animals (Scientific Procedures) Act 1986. Female Balb/c mice were infected intranasally with approximately 2 × 102 cfu of M. tuberculosis H37Rv (sample mean 2.13 × 102), and sacrificed 21 days post infection. Excised lung tissue was fixed with 4% paraformaldehyde for 24 hours and embedded in paraffin wax. 4 µm tissue sections were dewaxed, rehydrated, endogenous peroxidase activity blocked by incubation with 3% H2O2 in methanol and epitopes demasked with proteinase K. Samples were incubated with 1:50 pMLKL antibody in TBS; sequential sections were run with rabbit IgG as an isotype control. Samples were incubated with biotinylated horse anti-rabbit IgG and antibody binding was amplified using avidin-biotin-peroxidase conjugate. Samples were developed with NovaRed substrate (Vector Laboratories), and counter stained with Mayer's haematoxylin. Acid fast bacilli in lung sections were visualised using Ziehl-Neelsen staining.
Butler R.E., Krishnan N., Garcia-Jimenez W., Francis R., Martyn A., Mendum T., Felemban S., Locker N., Salguero F.J., Robertson B, & Stewart G.R. (2017). Susceptibility of Mycobacterium tuberculosis-infected host cells to phospho-MLKL driven necroptosis is dependent on cell type and presence of TNFα. Virulence, 8(8), 1820-1832.
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10

Immunohistochemical Analysis of Murine Tissues

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Paraffin-embedded sections were prepared as previously described (Haffner-Luntzer et al., 2016 (link)). To perform antigen retrieval, slides with sections were immersed in 10 mM citrate buffer (pH 6.0) in a 95°C water bath for 20 min. The sections were incubated with primary antibodies (rabbit polyclonal anti-mouse p16INK4a antibody, 1:1000, ab189034, Abcam, Cambridge, UK; rabbit monoclonal anti-mouse FoxO1 antibody, 1:1000, 2880, Cell Signaling, Danvers, MA, USA; rabbit polyclonal anti-mouse TNF-α antibody, 1:100, ab6671, Abcam; rabbit polyclonal anti-mouse IL-6 antibody, 1:250, bs-0782R, Bioss, Woburn, MA, USA), and with the secondary goat anti-rabbit antibody, followed by incubation with avidin-biotin complex and NovaRed substrate (Vector, Peterborough, UK), or with secondary goat anti-rabbit Alexa Fluor 594 (Thermo Fisher Scientific). Control sections were treated in parallel and incubated with the isotype control antibody (011-000-003, Jackson Immunoresearch, Ely, UK). For nuclei staining, Hematoxylin (Waldeck, Münster, Germany) was used. Before analyzing sections microscopically, the slides were mounted with Vitro-Clud (Langenbrinck, Emmendingen, Germany) or Fluoromount (Sigma Aldrich, Taufkirchen, Germany).
Schoppa A.M., Chen X., Ramge J.M., Vikman A., Fischer V., Haffner-Luntzer M., Riegger J., Tuckermann J., Scharffetter-Kochanek K, & Ignatius A. (2022). Osteoblast lineage Sod2 deficiency leads to an osteoporosis-like phenotype in mice. Disease Models & Mechanisms, 15(5), dmm049392.
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