Longitudinal sections (5 µm thick) from the control and tumor-bearing tibiae were deparaffinized and examined by immunohistochemistry for expression and localization of BIP (1:200), survivin (1:100), and CAIX (1:50). Human bone biopsy samples were decalcified, paraffin embedded, deparafinized, and cut into 5 µm sections. Immunohistochemical staining was performed to determine expression of localization of CK18 (1:50) and HO-1(1:100). For both xenograft and patient samples ImmPRESS Anti-Goat Peroxidase Polymer Detection systems along with a NovaRED kit (Vector Labs, Burlingame, CA) as a substrate were used for the peroxidase-mediated immunostaining reaction.
Novared kit
Manufactured by Vector Laboratories
Sourced in United States
The NovaRed kit is a laboratory product developed by Vector Laboratories. It is designed for the detection of peroxidase activity in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Halo image analysis platform, by Indica Labs (2 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Cd31 clone jc70a, by Agilent Technologies (1 mentions)
Streptavidin hrp complex, by Agilent Technologies (1 mentions)
Ventana discovery ultra system, by Roche (1 mentions)
Vector abc detection kit, by Vector Laboratories (1 mentions)
Entellan, by Merck Group (1 mentions)
Stereo investigator software, by MicroBrightField (1 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (1 mentions)
Retiga 2000r camera, by MicroBrightField (1 mentions)
γh2ax antibody, by Merck Group (1 mentions)
Antigen unmasking solution, by Vector Laboratories (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
Protein blocker, by Agilent Technologies (1 mentions)
Envision hrp, by Agilent Technologies (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Tenascin c, by Abcam (1 mentions)
Abc elite, by Vector Laboratories (1 mentions)
Power block, by BioGenex (1 mentions)
15 protocols using novared kit
1
Immunohistochemical Analysis of Bone Samples
2
Immunohistochemical Analysis of Vascular Markers
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Individual cross-sections were incubated with antibodies against aquaporin-1 (clone 1/A5F6, Abcam, Cambridge, UK), ICAM-1 (clone My13, Zymed, South San Francisco, CA), CD31 (clone JC70A, Dako, Glostrup, Denmark), monocyte/macrophage (clone HAM56, Dako), actin (smooth muscle)(clone 1A4, Dako). All antibodies were diluted in TBS containing 1% weight/volume (w/v) bovine serum albumin (BSA), (Sigma-Aldrich, Zwijndrecht, the Netherlands) and 0.01% (w/v) Tween-20 (Sigma-Aldrich). The incubations with secondary biotin-conjugated antibodies (Dako) were followed by amplification with a streptavidin-HRP complex (Dako), and a peroxidase-substrate staining (Nova Red kit, Vector Labs, Burlingame, CA).
3
Immune Cell Analysis in Coronary Atherosclerosis
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Human coronary artery autopsy samples with adjacent perivascular adipose tissue from an HIV-positive and an HIV-negative donor with a similar degree of atherosclerosis, and from two HIV-positive donors with and without a recorded diagnosis of diabetes prior to death, were obtained from CVPath Institute Registry for immunohistochemical staining as previously published.83 (link) Briefly, the artery segments were fixed in formalin, and 2 to 3 millimeter segments were embedded in paraffin. Sections of 5 microns thick were cut from each of the segments and mounted on slides. Immunohistochemistry for CD4, CX3CR1 and granzyme B were performed on the sections using Ventana DISCOVERY Ultra system (Roche). Slides were incubated with CX3CR1 antibody (Abcam ab8021, 1:1000 dilution), CD4 (Roche, 790-4423, pre-diluted) or anti-granzyme B (LifeSpan Biosciences LS-B7602) and developed by the NovaRed kit (Vector Laboratories). The images were captured by Axio Scan. Z1 (Zeiss, Germany) using a 20X objective, and images were processed and prepared on the HALO image analysis platform (Indica Labs, Corrales, NM).
4
Immunohistochemical Analysis of WNK1 in Tumor Tissue
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Briefly, 4-μm–thick sections of paraffin-embedded tissue samples were cut onto silane-treated Super Frost slides (CML, Nemours, France) and left to dry at 37°C overnight. Tumor sections were deparaffinized in xylene and rehydrated in pure ethanol. Before immunostaining, antigen retrieval was performed by immersing sections in citrate buffer, pH 6.0 (WNK1) (15 min at 95°C), washed twice in phosphate-buffered saline (PBS) for 3 minutes, and treated with 3% H2O2-PBS for 15 minutes to inhibit endogenous peroxidases. After washing in PBS, slides were saturated for 25 minutes in 3% bovine serum albumin PBS. Sections then were incubated for 1 hour at room temperature with antibody to WNK1 (dilution 1/100; clone ab128858; Abcam, Cambridge, United Kingdom). After washing in PBS, secondary antibody (8114P; Cell Signaling, Danvers, MA) was added for 30 minutes at room temperature. Slides were washed twice for 5 minutes in PBS and shown using the Novared kit (Vector, Burlingame, CA). Slides were washed twice in water for 5 minutes and counterstained with 10% Meyer's hematoxylin. After 1 wash in water, slides were dehydrated in 100% ethanol and in xylene for 30 seconds each. Apoptosis was quantified by counting the number of labeled cells with anti–caspase 3 antibody per 100 tumor cells in the most affected areas.
5
Immunohistochemical Analysis of Coronary Plaque
6
Immunohistochemical Analysis of Parkinson's Pathology
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Brains were extracted and post-fixed in 4% PFA for 24 h and sunk in 30% sucrose. Brains were frozen on a microtome platform and cut to generate 16 and 40 μm thick sections. A series of free-floating coronal sections was stained for either tyrosine hydroxylase (TH) or phosphorylated αSyn (pSyn). Tissue was incubated in 0.3% H2O2 for 45 min, rinsed, and blocked in 10% normal goat serum (1 h), then incubated in primary mouse anti-pSyn (Ser129) (1:500, BioLegend, San Diego, CA, USA), mouse-anti-TH (1:200) antibodies overnight at 4 °C. Then, sections were incubated in biotinylated secondary antisera against either mouse (1:400, Millipore, Temecula, CA, USA) or rabbit IgG (1:400, Millipore, Temecula, CA, USA) followed by the Vector ABC detection kit (Vector Labs, Burlingame, CA, USA). Antibody labeling was visualized by exposure to 0.5 mg/mL 3,3′ diaminobenzidine (DAB), 2.5 mg/mL nickel ammonium sulfate and 0.03% H2O2 in Tris buffer followed by incubation with the NovaRed kit (Vector Labs, Burlingame, CA, USA). Sections were mounted on subbed slides, dehydrated to xylene, and coverslipped with xylene base mounting buffer.
7
Immunohistochemistry of PARP-1 in FFPE
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For immunohistochemistry, FFPE histological sections were dewaxed, rehydrated in an ethanol series (95%, 85%, and 70%) and then subjected to microwave antigen retrieval in 0.01 M citrate (pH 6.0; 0.05% Tween-20) and methanol/H2O2 treatment. After blocking with 5% goat serum, the slides were sequentially incubated with anti-poly-ADP-ribose binding reagent (Millipore-Sigma, MABE1031) at a 1:1,000 dilution and HRP-labeled secondary antibodies. A NovaRed kit (Vector, SK-4800) was used for visualization.
8
Astrocyte and Microglia Activation in Spinal Cord
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To investigate the activation of astrocytes and microglia cells in the lumbar tract of the spinal cord, immunohistochemistry for light microscopy was performed. The sections were incubated for 10 min in 3% H2O2 to quench endogenous peroxidase and preincubated for 1 h in 5% of NGS in PBS and 1% BSA. The slides were incubated overnight in anti-mouse GFAP or Iba1 antibodies to recognize astrocytes and microglia, respectively, (GFAP 1:500, Z0334 Dako; Iba1 1:500 019–19741 Wako) in 1% NGS in PBS. The sections were washed and incubated for 1 h in biotinylated goat anti-rabbit IgG (1:100, Vector Laboratories). The avidin-biotin peroxidase kit (ABC kit; Vector) and Novared kit (Vector) as signal revelation system was used. After mounting on slides, the sections were dehydrated through increasing grades of ethanol, cleared in xylene, and coverslipped with Entellan (Merck, Darmstadt, Germany). For the analysis, cells were counted every 100 µm for a total of 30 sections for each animal. The astrocytes and microglial cells (GFAP or CD11b labeled cells) of the lumbar tract (L1-L5) were visualized and counted using a computer-assisted microscope (Olympus BX6 with Retiga 2000R camera) with the Stereoinvestigator software (MicroBrightField, Williston, VT, USA).
9
Immunohistochemical Staining Protocol
10
Immunohistochemical Analysis of γH2AX in FFPE Tissue
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For immunohistochemistry, human FFPE histological sections were dewaxed, rehydrated in an ethanol series, and then subjected to microwave antigen retrieval in 0.01 M citrate (pH 6.0) and methanol/H2O2 treatment. After blocking with 5% goat serum, the slides were sequentially incubated with γH2AX antibody (Sigma-Aldrich, 05-636, clone JBW301) at a 1:1,000 dilution and HRP-labeled secondary antibodies. A NovaRed kit (Vector, SK-4800) was used for visualization.
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