Formalin-fixed paraffin embedded (FFPE) sections (5 μm) containing left upper and lower lung lobes were cleared in Histoclear (3 × 5 min) (National Diagnostics). Sections were rehydrated with graded alcohols as follows: 100% ethanol (2 × 5 min), 95% ethanol (1 × 5 min), 70% ethanol (1 × 5 min), and H2O (1 × 5 min). To inactivate endogenous peroxidases, rehydrated sections were immersed in 3% H2O2/70% methanol solution (Fisher Scientific) for 30 min. Sections were incubated with biotin-conjugated HHL (1:300; Vector Laboratories) overnight at 4°c . After washing in PBS, sections were incubated with an avidin/biotin-based peroxidase system (VECTASTAIN Elite ABC HRP Kit; Vector Laboratories) followed by substrate deposition with VECTOR NovaRED Peroxidase (HRP) Substrate Kit (Vector Laboratories). Nuclear counterstain was performed with Gill’s Hematoxylin (American MasterTech Scientific) followed by mounting with Permount (Fisher Scientific).
Vector novared peroxidase hrp substrate kit
Manufactured by Vector Laboratories
Sourced in United States
The VECTOR NovaRED Peroxidase (HRP) Substrate Kit is a laboratory reagent used for the detection and visualization of enzyme-labeled antibodies or other proteins in immunohistochemical and related applications. The kit provides a chromogenic substrate that is oxidized by the presence of the horseradish peroxidase (HRP) enzyme, resulting in the formation of a red-colored reaction product.
Automatically generated - may contain errors
Lab products found in correlation
Vectastain elite abc hrp kit, by Vector Laboratories (3 mentions)
Goat serum, by Abcam (2 mentions)
Mouse anti brdu monoclonal antibody, by BD (2 mentions)
Harris haematoxylin solution, by Merck Group (2 mentions)
Histoclear, by National Diagnostics (1 mentions)
Permount, by Thermo Fisher Scientific (1 mentions)
Evos xl core cell imaging system, by Thermo Fisher Scientific (1 mentions)
Netrin 1 rat anti mouse monoclonal antibody clone, by R&D Systems (1 mentions)
Benchmark xt, by Roche (1 mentions)
Clone c4, by Santa Cruz Biotechnology (1 mentions)
F4 80, by Santa Cruz Biotechnology (1 mentions)
Streptavidin horseradish peroxidase hrp, by Vector Laboratories (1 mentions)
Dp72 camera, by Olympus (1 mentions)
Cellsens software, by Olympus (1 mentions)
Anti casp3, by Cell Signaling Technology (1 mentions)
Anti g6pd, by Abcam (1 mentions)
Anti glul, by Merck Group (1 mentions)
Anti gls, by Abcam (1 mentions)
Anti cdkn1a, by Abcam (1 mentions)
Dako envision system hrp labelled polymer, by Agilent Technologies (1 mentions)
17 protocols using vector novared peroxidase hrp substrate kit
1
Lung Immunohistochemistry Protocol
2
Immunohistochemical Liver Cell Proliferation
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Fresh liver tissues were fixed in 10% buffered formalin, embedded in paraffin, and sections were prepared and stained by the Connecticut Veterinary Medical Diagnostic Laboratory at the University of Connecticut (Storrs, CT, USA). Immunohistochemistry was performed using antibodies against Ki-67 (rabbit polyclonal, Cat. No. ab15580) and proliferating cell nuclear antigen (PCNA) (rabbit polyclonal, Cat. No. ab18197) from Abcam (Cambridge, UK). A secondary antibody conjugated to peroxidase allowed for color precipitation using a VECTOR NovaRED Peroxidase (HRP) Substrate Kit from Vector Laboratories (Burlingame, CA, USA). Images were captured using an EVOS XL Core Cell Imaging System from Thermo Fisher Scientific (Waltham, MA, USA). Positively stained nuclei were identified by using the ImageJ image processing program, version 1.50i, from National Institutes of Health (Bethesda, MD, USA) using a color brightness threshold with a signal intensity greater than or equal to 200.
3
Netrin-1 Expression in Lymphoma: Immunohistochemical Evaluation
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Immunohistochemical staining for netrin-1 (169 nodal lymphomas and 32 normal lymph nodes) was performed in a manual way, with the avidin-biotin-peroxidase complex method, on formalin-fixed, paraffin-embedded tissues. Netrin-1 expression was evaluated using a netrin-1 rat anti-mouse monoclonal antibody (clone, diluted 1:100, R&D Systems, Minneapolis, MN, USA).
Antigen retrieval was carried out at 95 °C for 40 min in a pH9 solution (Dako target retrieval solution, pH 9, Dako, Carpinteria, CA, USA). Amplification of the labeling was performed by the ultraTek HRP (anti-polyvalent) ready-to-use kit (ScyTek Laboratories, Logan, UT, USA) 30 min at 20 °C and revelation with the Vector NovaRED Peroxidase (HRP) Substrate kit (Vector, Burlingame, CA, USA) for 5 min. After a 3 min counterstaining with Hematoxylin, sections were dehydrated and mounted. For each sample, a negative control was realized, by replacing the anti-netrin-1 antibody with the antibody diluent solution (Emerald diluent antibody, Cellmarque, Sigma Aldrich, CA, USA).
A qualitative and semi-quantitative evaluation of netrin-1 expression was performed. The type of immunostained cell and the staining location were recorded. Semi-quantitative grading of positively labelled lymphoma cells was realized, according toTable 1 .
Antigen retrieval was carried out at 95 °C for 40 min in a pH9 solution (Dako target retrieval solution, pH 9, Dako, Carpinteria, CA, USA). Amplification of the labeling was performed by the ultraTek HRP (anti-polyvalent) ready-to-use kit (ScyTek Laboratories, Logan, UT, USA) 30 min at 20 °C and revelation with the Vector NovaRED Peroxidase (HRP) Substrate kit (Vector, Burlingame, CA, USA) for 5 min. After a 3 min counterstaining with Hematoxylin, sections were dehydrated and mounted. For each sample, a negative control was realized, by replacing the anti-netrin-1 antibody with the antibody diluent solution (Emerald diluent antibody, Cellmarque, Sigma Aldrich, CA, USA).
A qualitative and semi-quantitative evaluation of netrin-1 expression was performed. The type of immunostained cell and the staining location were recorded. Semi-quantitative grading of positively labelled lymphoma cells was realized, according to
4
Automated and Manual BAP1 Immunohistochemistry
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Immunohistochemistry was performed on 4-μm-thick FFPE sections using an automated immunostainer (Benchmark XT, Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s instructions. Sections were immunostained with a primary monoclonal antibody against BAP1 (clone C4, 1:100 dilution, Santa Cruz Biotechnology, Dallas, TX, USA) using 3,3-diaminobenzidine (DAB) as chromogen. Selected cases that showed a staining result that was difficult to interpret including strongly pigmented tumors (patients #1–8, #12–16, #22–24) were manually stained (clone C4, 1:50 dilution, Immunologic, Duiven, the Netherlands) according to the manufacturer’s instructions. The VECTOR NovaRED Peroxidase (HRP) Substrate Kit was used for visualization (Vector Laboratories, USA, Catalogue Number SK-4800). Nuclei of endothelial and lymphocytic cells in the slides served as positive internal control for BAP1 protein expression. The staining results were scored independently by three pathologists (HK, DC, WB). The percentage of positive tumor cell nuclei was scored only in areas with positive internal controls.
5
Adipocyte Morphometry and Macrophage Quantification
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Liver and perigonadal visceral adipose tissue (VAT) was removed, fixed and processed to paraffin blocks. Tissue sections were stained with haematoxylin and eosin (H & E). Adipocyte area and diameter were measured using H & E-stained VAT sections in 9 to 10 fields at 200 -power fields using cellSens software (Olympus, Tokyo, Japan). Adipocyte numbers were determined in 5 fields at 200 -power field. For Oil Red O staining, frozen liver sections were prepared using O.C.T. compound (Tissue-Tek) prior to staining (UHN Pathology Research Program, Toronto, ON, Canada).
Adipose tissue macrophages in VAT sections were detected by immunostaining for F4/80 (1:200 dilution, Santa Cruz Biotechnology) for 2 hours at room temperature (RT). Primary antibodies were detected using goat anti-rat IgG-biotinylated (1:200 dilution, Santa Cruz Biotechnology) with 30 minutes incubation at RT. Secondary antibodies were detected using streptavidin horseradish peroxidase (HRP) (Vector labs) for 30 minutes at RT followed by colour development Vector NovaRED Peroxidase (HRP) Substrate Kit (Vector labs) as per manufacturer’s instructions. CLS were quantified per 100 -power field in at least 15 fields in a blinded manner using Leica DM1000 microscope (Wetzlar, Germany) with Olympus DP72 camera (Waltham, MA, USA).
Adipose tissue macrophages in VAT sections were detected by immunostaining for F4/80 (1:200 dilution, Santa Cruz Biotechnology) for 2 hours at room temperature (RT). Primary antibodies were detected using goat anti-rat IgG-biotinylated (1:200 dilution, Santa Cruz Biotechnology) with 30 minutes incubation at RT. Secondary antibodies were detected using streptavidin horseradish peroxidase (HRP) (Vector labs) for 30 minutes at RT followed by colour development Vector NovaRED Peroxidase (HRP) Substrate Kit (Vector labs) as per manufacturer’s instructions. CLS were quantified per 100 -power field in at least 15 fields in a blinded manner using Leica DM1000 microscope (Wetzlar, Germany) with Olympus DP72 camera (Waltham, MA, USA).
6
Immunohistochemical Analysis of Metabolic Proteins
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Four-micron-thick paraffin-embedded sections were collected and stained for H&E and were incubated overnight with the following antibodies: anti-c-Myc (Abcam, Cambridge, United Kingdom; ab32072), anti-KI-67 (Abcam, ab15580), anti-G6PD (Abcam, ab87230), anti-GLUL (Sigma-Aldrich G2781), anti-GLS (Abcam, ab 262716), and anti-CASP3 (Cell Signaling Technology, Danvers, MA; 9664L), and anti-CDKN1A (Abcam, ab107099). The secondary antibodies anti-rabbit Dako EnVision+ System Labelled Polymer-HRP (Dako, Carpinteria, CA) or goat anti-rabbit Biotinylated IgG Antibody (H+L) (Vector Laboratories, Newark, CA; BA-1000) were used. Peroxidase binding sites were detected by Vector NovaRED Peroxidase (HRP) Substrate Kit (Vector Laboratories).
7
Kidney Tissue Immunohistochemistry Protocol
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Kidneys were fixed in 4% paraformaldehyde overnight and then embedded in paraffin. Kidney tissue sections were deparaffinized by three changes of Histoclear II (National Diagnostics, Atlanta, GA, USA) and then rehydrated in a graded series of ethanol. For antigen retrieval, the sections were heated in a TintoRetriever Pressure Cooker (Bio SB, Santa Barbara, CA, USA) with Borg Decloaker RTU (Biocare Medical, Pacheco, CA, USA) and were incubated with UV Hydrogen Peroxide Block (Thermo Fisher Scientific, Waltham, MA, USA). Sections were blocked with blocking solution for 1 h at room temperature, and then incubated overnight with the appropriate primary antibodies at 4°C. The primary antibodies used for immunostaining were anti-8-OHdG antibody (sc-66036, Santa Cruz) and anti-4 hydroxynonenal (ab48506, Abcam). Peroxidase activity was detected using the VECTASTAIN Elite ABC HRP Kit (PK-6200, Vector Labs, Burlingame, CA, USA) and VECTOR NovaRED Peroxidase (HRP) Substrate Kit (SK-4800, Vector Labs) according to the manufacturer’s instructions. Slides were mounted with VectaMount Permanent Mounting Medium (H-5000, Vector Labs) and analysed.
8
Immunohistochemical Assessment of Cell Proliferation
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Liver, pancreas, kidney and heart sections were fixed in 10% of buffered formalin and processed for staining with haematoxylin‐eosin (H&E) or immunohistochemistry (IHC). For BrdU detection, paraffin‐embedded 4 µm sections were deparaffinized, treated with HCl 2N for 1h and then with 0,1% trypsin at 37°C. Sections were sequentially incubated with goat serum (Abcam), mouse monoclonal anti‐BrdU antibody (Becton Dickinson, San Jose, CA) and with Dako EnVision+® System Labelled Polymer‐HRP anti‐mouse (Dako Corporation, Carpinteria, CA). Peroxidase binding sites were detected by Vector Novared Peroxidase (HRP) Substrate Kit (Vector Labs, Burlingame, CA). Harris haematoxylin solution (Sigma) was used to counterstain liver sections. Labelling index (LI) was expressed as the number of BrdU‐positive hepatocyte nuclei/100 nuclei. Mitotic index was calculated as the number of mitotic figures/1000 nuclei. Results were expressed as the means ± SD of 3 to 5 animals per group. At least 3000 hepatocyte nuclei per liver were scored.
9
Immunohistochemical Analysis of Pancreatic Markers
10
Immunohistochemical Staining of Mouse Lung
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Formalin-fixed paraffin-embedded mouse lung tissue was used for staining with AOX antiserum (1:2000; custom raised in rabbit; 21st Century Biochemicals, Marlborough, MA, USA) (42 (link)), visualized using the ZytoChem Plus (HRP) Polymer Bulk Kit (Zytomed Systems GmbH, Berlin, Germany) and VECTOR NovaRED Peroxidase (HRP) Substrate Kit (Vector Laboratories, Burlingame, USA).
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