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3 3 diaminobenzidine dab detection kit

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The 3,3'-diaminobenzidine (DAB) detection kit is a laboratory product used for the detection of specific target proteins in biological samples. The kit provides a chromogenic substrate that produces a brown reaction product when catalyzed by the enzyme horseradish peroxidase (HRP), which is commonly used as a labeling agent in immunohistochemistry and other protein detection techniques.

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Lab products found in correlation

Discovery xt processor, by Roche (6 mentions) Permount, by Thermo Fisher Scientific (5 mentions) N68 6, by Abcam (2 mentions) Ventana benchmark ultra, by Roche (1 mentions) Biotinylated goat anti rabbit igg, by Vector Laboratories (1 mentions) Bond polymer refine detection kit, by Leica (1 mentions) Nb100 479, by Novus Biologicals (1 mentions)

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IVIEW 3,3-diaminobenzidine (DAB) detection kit 3–3′-diaminobenzidine detection kit DAB DAB detection kit Diaminobenzidine (DAB) DAB kit

8 protocols using 3 3 diaminobenzidine dab detection kit

1

Automated Immunohistochemistry Protocol

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Immunohistochemistry (IHC) was performed using a BenchMark automated staining instrument (Ventana Medical System, Tucson, AZ, USA). Primary antibodies used for IHC staining are listed in Supplementary Table S3. Tissue samples were sectioned to 4 µm thickness, deparaffinized in xylene, rehydrated in three graded alcohol chambers, and treated with 3% hydrogen peroxide in methanol. For stain visualization, a 3,3′-Diaminobenzidine (DAB) detection kit (Ventana Medical System, Tucson, AZ, USA) was used.
Kim H.M, & Koo J.S. (2020). Clinicopathologic Characteristics of Breast Cancer According to the Infiltrating Immune Cell Subtypes. International Journal of Molecular Sciences, 21(12), 4438.
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2

Immunohistochemical Analysis of Nav1.7 Expression in Mouse Sciatic Nerve

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Immunohistochemical (IHC) staining experiments were used to detect the expression and abundance of sodium channel NaV1.7 in mouse sciatic nerve tissue. Anti-NaV1.7 antibody [N68/6] (Abcam ab85015) was found to specifically bind to mouse NaV1.7 (0.5 μg/mL). Paraffin-embedded formalin-fixed 5 μm sections were deparaffinized with EZPrep buffer. For IHC detection, a 3,3′-diaminobenzidine (DAB) detection kit (Ventana Medical Systems, Tucson, AZ) was used according to the manufacturer’s instructions. These experiments were performed at the MSKCC Molecular Cytology Core Facility using the Discovery XT processor (Ventana Medical System, Tucson, AZ). Adjacent sections were stained against IgG, to control for non-specific binding to NaV1.7. Sections were counterstained with hematoxylin and eosin (H&E) and coverslipped with Permount (Fisher Scientific, Pittsburgh, PA) for morphological evaluation of tissue characteristics.
Gonzales J., Pirovano G., Chow C.Y., de Souza Franca P.D., Carter L.M., Klint J.K., Guru N., Lewis J.S., King G.F, & Reiner T. (2020). Fluorescence labeling of a NaV1.7-targeted peptide for near-infrared nerve visualization. EJNMMI Research, 10, 49.
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3

Automated Immunohistochemical Staining Protocol

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All the immunohistochemistry experiments were performed on a Ventana Benchmark Ultra automated staining (Ventana Medical Systems Inc., Tuscon, AZ, U.S). Immunohistochemistry of the tumor samples was performed on 4 μm sections. The settings included pre-treatment with cell conditioner 1 (CC1) buffer for 56 min, incubation with a phosphorylated STAT3 (1:200) antibody for 40 min, and pre-treatment with CC1 buffer for 76 min, incubation with a phosphorylated YAP1 (1:200) antibody for 28 min. The detection was performed with 3,3-diaminobenzidine (DAB) detection kit (Ventana Medical Systems Inc.) according to the manufacturer's instructions. Slides were counterstained with hematoxylin and mounted. The evaluation of immunohistochemically labeled samples with these antibodies included the percentage of positively stained tumor cells and their intensity as well as the staining localization (cytoplasmic, nuclear). The semi-quantitative evaluation was performed using the H-score classification. The percentage of negative (0), weakly stained (1+), moderately stained (2+) and strongly stained (3+), tumor cells was estimated and the H-score was calculated as follows: H-score = 1 × (% of 1+ staining) + 2 × (% of 2+ staining) + 3 × (% of 3+ staining).
Karachaliou N., Chaib I., Cardona A.F., Berenguer J., Bracht J.W., Yang J., Cai X., Wang Z., Hu C., Drozdowskyj A., Servat C.C., Servat J.C., Ito M., Attili I., Aldeguer E., Capitan A.G., Rodriguez J., Rojas L., Viteri S., Molina-Vila M.A., Ou S.H., Okada M., Mok T.S., Bivona T.G., Ono M., Cui J., Cajal S.R., Frias A., Cao P, & Rosell R. (2018). Common Co-activation of AXL and CDCP1 in EGFR-mutation-positive Non-Small Cell Lung Cancer Associated With Poor Prognosis. EBioMedicine, 29, 112-127.
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4

Immunohistochemical Detection of NaV1.7 in Nerves

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Nav1.7 in human vagus nerves and mouse sciatic nerves was detected using immunohistochemical (IHC) staining techniques, which were performed at the Molecular Cytology Core Facility of MSK using the Discovery XT processor (Ventana Medical System, Tucson, AZ). Anti-Nav1.7 antibody [N68/6] (Abcam ab85015) specifically bound to both human and mouse Nav1.7 (0.5 μg/mL). Paraffin-embedded formalin-fixed 10 μm sections were deparaffinized with EZPrep buffer. For IHC detection, a 3,3′-diaminobenzidine (DAB) detection kit (Ventana Medical Systems, Tucson, AZ) was used according to the manufacturer’s instructions. Sections were counterstained with H&E and coverslipped with Permount (Fisher Scientific, Pittsburgh, PA).
Gonzales J., de Souza Franca P.D., Jiang Y., Pirovano G., Kossatz S., Guru N., Yarilin D., Agwa A.J., Schroeder C.I., Patel S.G., Ganly I., King G.F, & Reiner T. (2019). Fluorescence Imaging of Peripheral Nerves by a Nav1.7-Targeted Inhibitor Cystine Knot Peptide. Bioconjugate chemistry, 30(11), 2879-2888.
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5

Immunohistochemical Analysis of Tumor Markers

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Tumor tissue samples were fixed overnight in 4% paraformaldehyde, embedded in paraffin, then sectioned. Immunohistochemistry for C/EBPα was performed using Bond Polymer Refine detection kit (Leica) according to the manufacturer’s instructions, and that for Ki-67, cleaved caspase 3, and HIF1α using Discovery XT processor (Ventana Medical Systems). Slides were heated for 30 min for antigen retrieval and incubated with either anti-C/EBPα (Cell Signaling 8178) for 30 min, anti-Ki-67 (Abcam ab15580, 0.1 μg/mL) for 4 h, or anti-cleaved caspase 3 (Cell Signaling 9661, 0.1 μg/mL) for 3 h, and anti-HIF1α (Novus Bio NB100-479) for 5 h (all antibodies are rabbit polyclonal). After washing, slides were incubated for 20 min with biotinylated goat anti-rabbit IgG (Vector Labs PK6101, 5.75 μg/mL), blocked with Blocker D, and staining detected using a 3,3’-diaminobenzidine (DAB) detection kit (Ventana Medical Systems) according to the manufacturer’s instructions. Slides were counterstained with hematoxylin and cover-slipped with Permount (Fisher Scientific). Whole stained slides were scanned, and 4–11 randomly selected representative fields containing ≥ 100 nuclei were counted to determine the ratio of cells with C/EBPα or Ki67 nuclear staining relative to all nuclei by manual counting or using Image J (NIH).
Angeles C.V., Velez A., Rios J., Laxa B., Shum D., Ruiz P.D., Shen Y., Ostrovnaya I., Gularte-Mérida R., Nacev B.A., Dickson M.A., Djaballah H., Okada T, & Singer S. (2021). A High-Content Screen for C/EBPα Expression Identifies Novel Therapeutic Agents in Dedifferentiated Liposarcoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 28(1), 175-186.
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6

Immunohistochemical Quantification of NaV1.7

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NaV1.7 staining was performed at the Molecular Cytology Core Facility of MSK using a Discovery XT processor (Ventana Medical System, Tucson, AZ). We used an anti-NaV1.7 antibody [N68/6] (NeuroMab) that binds to both human and mouse NaV1.7 (0.5 μg/mL). Paraffin-embedded formalin-fixed 4 μm sections were deparaffinized with EZPrep buffer. For IHC, a 3,3′-diaminobenzidine (DAB) detection kit (Ventana Medical Systems, Tucson, AZ) was used according to the manufacturer’s instructions. Sections were counterstained with hematoxylin and eosin (H&E) and coverslip using Permount (Fisher Scientific, Pittsburgh, PA).
NaV1.7 quantification was performed on digitalized slides. The threshold for signal intensity in the DAB (brown) and H&E (blue, representing all tissue area) channels was determined via an automated script using ImageJ analysis software. Color deconvolution was used to separate blue and brown signals and the threshold values were kept constant: 0–114 for DAB and 0–235 for H&E. The relative NaV1.7 positive area was calculated by dividing the brown (DAB) area by the blue (total tissue area).
Adilbay D., Gonzales J., Zazhytska M., Demetrio de Souza Franca P., Roberts S., Viray T., Artschwager R., Patel S., Kodra A., Overdevest J.B., Chow C.Y., King G.F., Jain S.K., Ordonez A.A., Carroll L.S., Reiner T, & Pillarsetty N. (2022). Non-invasive diagnostic method to objectively measure olfaction and diagnose smell disorders by molecularly targeted fluorescent imaging agent. bioRxiv.
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7

Immunohistochemical Analysis of NaV1.7 in Human Vagus Nerves

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Nerves donated by the Fusion Solution Bioskills Laboratory autopsy program were used in immunohistochemistry experiments. NaV1.7 in human vagus nerves was detected using immunohistochemical (IHC) staining techniques at the Molecular Cytology Core Facility of MSK using the Discovery XT processor (Ventana Medical System, Tucson, AZ). Anti-NaV1.7 antibody [N68/6] (Abcam ab85015) specifically bound to both human and mouse NaV1.7 (0.5 μg/mL). Paraffin-embedded formalin-fixed 10 μm sections were deparaffinized with EZPrep buffer. For IHC detection, a 3,3′-diaminobenzidine (DAB) detection kit (Ventana Medical Systems, Tucson, AZ) was used according to the manufacturer’s instructions. In addition, sections were counterstained with hematoxylin and eosin (H&E) staining techniques and coverslipped with Permount (Fisher Scientific, Pittsburgh, PA).
Gonzales J., Hernández-Gil J., Wilson T.C., Adilbay D., Cornejo M., de Souza Franca P.D., Guru N., Schroeder C.I., King G.F., Lewis J.S, & Reiner T. (2021). Bimodal Imaging of Mouse Peripheral Nerves with Chlorin Tracers. Molecular pharmaceutics, 18(3), 940-951.
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8

Immunohistochemical Detection of Nav1.7 in Mouse Sciatic Nerve

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Immunohistochemical (IHC) staining experiments were used to detect the expression and abundance of sodium channel Na V 1.7 in mouse sciatic nerve tissue. Anti-Na V 1.7 antibody [N68/6] (Abcam ab85015) was found to speci cally bind to mouse Na V 1.7 (0.5 µg/mL). Para n-embedded formalin-xed 5 µm sections were depara nized with EZPrep buffer. For IHC detection, a 3,3'-diaminobenzidine (DAB) detection kit (Ventana Medical Systems, Tucson, AZ) was used according to the manufacturer's instructions. These experiments were performed at the MSKCC Molecular Cytology Core Facility using the Discovery XT processor (Ventana Medical System, Tucson, AZ). Adjacent sections were stained against IgG, to control for non-speci c binding to Na V 1.7. Sections were counterstained with hematoxylin and eosin (H&E) and coverslipped with Permount (Fisher Scienti c, Pittsburgh, PA) for morphological evaluation of tissue characteristics.
Gonzales J., Pirovano G., Chow C.Y., França P.D.D.S., Carter L.M., Klint J.K., Guru N., Lewis J.S., King G.F., & Reiner T. (2020). Fluorescence labeling of a NaV1.7-targeted peptide for near-infrared nerve visualization.
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