Dab kit

Manufactured by Vector Laboratories

Sourced in United States, United Kingdom, Canada

The DAB kit is a laboratory product designed for the detection and visualization of protein targets in biological samples. It provides a peroxidase-based chromogenic detection system that generates a brown reaction product upon interaction with the target protein. The kit contains the necessary reagents and solutions to perform this detection process.

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Lab products found in correlation

218 protocols using dab kit

Immunohistochemical Analysis of α-SMA and CD45

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The pancreatic paraffin sections were prepared for immunohistochemistry analysis of α-SMA and CD45 with ABC kit and DAB kit (Vector Laboratories, Inc. Burlingame, CA, USA) according to the manufacturer’s instructions as described previously [21] (link). Briefly, following deparaffinization and hydration, antigen retrieval, blockage of endogenous peroxidase activity and nonspecific protein binding sites, the sections were then incubated with rabbit antibodies against α-SMA and CD45 overnight at 4°C. The sections were subsequently incubated with a biotinylated anti-rabbit antibody for one hour at room temperature, and then with ABC reagents for 30 minutes at room temperature. Finally the sections were stained using DAB kit followed by hematoxylin nuclei counterstaining and dehydrated, mounted with a permanent mounting solution (Vector Laboratories, Inc., Burlingame, CA, USA).

Gao X., Cao Y., Staloch D.A., Gonzales M.A., Aronson J.F., Chao C., Hellmich M.R, & Ko T.C. (2014). Bone Morphogenetic Protein Signaling Protects against Cerulein-Induced Pancreatic Fibrosis. PLoS ONE, 9(2), e89114.

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Quantifying Dopaminergic Neurons in Mouse Brain

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Using the previously collected mouse brain samples, 35 µm coronal sections were cut (Leica CM1950), and free floating sections were incubated overnight at room temperature with rabbit anti-TH primary antibody (Novus Cat. NB300-109), followed by incubation for 1 h with secondary antibody using the Elite rabbit IgG kit (vector laboratory PK-6101) [43 (link)]. The DAB kit (Vector laboratory PK-4100) was used for color development.
The number of TH positive dopamine neurons in the SN was estimated using unbiased stereological counting. The SN was delineated from 1.70 to 3.88 mm posterior to bregma using the Allen brain atlas [44 (link)]. A total of 7 coronal sections from the midbrain of each mouse were used for quantification (1 section from every 5 serial coronal sections across the midbrain was used); representative pictures containing TH⁺ neurons in the SN were taken using the 10 × magnification objective lens of an Olympus IX83 microscope. Scale bar, 500 µm. The number of TH⁺ neurons in each section was stereologically quantified using Stereologer 2000™, at 63 × magnification. The 7 sections from each mouse were then used to compare the number of TH⁺ neurons in Tg and NTg mice (n = 5 for Tg, n = 5 for NTg).

Vanan S., Zeng X., Chia S.Y., Varnäs K., Jiang M., Zhang K., Saw W.T., Padmanabhan P., Yu W.P., Zhou Z.D., Halldin C., Gulyás B., Tan E.K, & Zeng L. (2020). Altered striatal dopamine levels in Parkinson’s disease VPS35 D620N mutant transgenic aged mice. Molecular Brain, 13, 164.

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Immunohistochemistry Protocol for Tissue Sections

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IHC was performed as previously described ^{3 (link)}. Tissue sections were treated by boiling in 0.01 M Citrate buffer (pH 6.0) for antigen retrieval, blocked in 5% normal goat serum, and incubated with primary antibodies diluted in 1% normal goat serum at 4°C overnight (Supplemental Table 2 for the antibody information). Slides were incubated with biotinylated secondary antibodies for 1 hour then with horseradish peroxidase streptavidin (SA-5004, Vector Laboratories, Burlingame, CA, USA) for 30 min, visualized by DAB kit (SK-4100, Vector Laboratories), then counterstained with 5% (w/v) Harris Hematoxylin, and subsequently mounted with Permount Mounting Medium (SP15–500, Thermo Fisher Scientific, Waltham, MA, USA).

He Y., Johnson D.T., Yang J.S., Wu H., You S., Yoon J., Lee D.H., Kim W.K., Aldahl J., Le V., Hooker E., Yu E.J., Geardts J., Cardiff R.D, & Sun Z. (2019). LOSS OF THE TUMOR SUPPRESSOR, TP53, ENHANCES THE ANDROGEN RECEPTOR MEDIATED ONCOGENIC TRANSFORMATION AND TUMOR DEVELOPMENT IN THE MOUSE PROSTATE. Oncogene, 38(38), 6507-6520.

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Aromatase Immunohistochemistry in Midbrain Sections

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Four midbrain sections containing the dorsal raphe were removed from −20 °C storage in cryoprotectant buffer and washed in KPBS buffer four times for 15 min each (rinsed), immersed in 1% hydrogen peroxide for 30 min, rinsed and then incubated with the following blocking solutions: Vector normal goat serum (NGS; Vector Laboratories, Burlingame, CA, USA) for 60 min; Vector avidin for 20 min; Vector biotin for 20 min. Sections were then incubated for 24 h at 4 °C in primary antibody to aromatase (Rabbit anti-human aromatase; polyclonal aa400–502; LS-B521; LifeSpan BioSciences, Inc., Seattle, WA, USA), This antibody was characterized across a range of titers with positive and negative controls. The primary antibody to aromatase was diluted in 2% NGS, 0.02 M KPBS and 0.4% Triton at 1/500. Sections were then rinsed, incubated in Vector biotinylated goat anti-rabbit serum for 60 min, rinsed, incubated with Vector ABC reagent for 60 min, rinsed, incubated with 0.05% diaminobenzidine (DAB kit, Vector Laboratories) containing 3% hydrogen peroxide for 1–10 min, and finally rinsed. Sections were mounted on Superfrost Plus slides (Thermo Fisher Scientific Inc., Waltham, MA, USA) and dried overnight under vacuum. The sections were further dehydrated through a graded series of ethanols and xylene. The sections were finally mounted under glass with DPX.

Bethea C.L., Phu K., Kim A, & Reddy A.P. (2015). ANDROGEN METABOLITES IMPACT CSF AMINES AND AXONAL SEROTONIN VIA MAO-A AND -B IN MALE MACAQUES. Neuroscience, 301, 576-589.

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Tissue Microarray Immunohistochemistry for p53

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The construction and analysis of tissue microarray containing all these human samples has been reported elsewhere^{4 (link),47 (link)}. At least two representative duplicate cores for each case were scored. Inmunohistochemistry analysis was carried out using the monoclonal mouse anti-human p53 (DO07, Agilent-Dako). Signal was amplified using avidin-peroxidase (ABC Elite Kit; Vector Labs), and peroxidase was visualized using 3,30-diaminobenzidine as a substrate (DAB kit, Vector Labs). Negative control slides were obtained by replacing primary antibodies with PBS (data not shown). Scoring of the results and selection of the thresholds, internal controls for reactivity of each antibody, and tissue controls for the series were done according to previously published methods^{4 (link),47 (link)}.

Dueñas M., Pérez-Figueroa A., Oliveira C., Suárez-Cabrera C., Sousa A., Oliveira P., Villacampa F., Paramio J.M, & Martínez-Fernández M. (2019). Gene Expression Analyses in Non Muscle Invasive Bladder Cancer Reveals a Role for Alternative Splicing and Tp53 Status. Scientific Reports, 9, 10362.

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Immunohistochemical Analysis of GATAD2A in Thyroid Cancer

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All tissues were fixed overnight in a formalin solution, dehydrated in ethanol, embedded in paraffin, and sectioned at 5 μm. Then, the slides were treated with xylene and ethanol to remove the paraffin. The slides were blocked with 5% normal goat serum and incubated with anti-GATAD2A overnight at 4 °C. After washing with PBS, the slides were incubated with goat anti-rabbit horseradish peroxidase (Jackson ImmunoResearch, PA, USA) for 1 h at room temperature, and was detected the immunohistochemical reactions using a DAB kit (Vector Laboratories, CA, USA). The slides were examined under a phase contrast light microscope (Olympus IX73, Japan), and the average integral optical density of each positively stained slide was measured using an Image-Pro plus 6.0 image analysis system. Three areas were chosen randomly from each section for measurement.
For validation of differentially expressed genes, the IHC data of thyroid cancer tissues and normal thyroid tissues were downloaded from Human Protein Atlas [26 (link)] available from www.proteinatlas.org. Protein expression levels were scaled based on an overall antibody staining score ranging from not detected to high.

Yao Y., Chen X., Yang H., Chen W., Qian Y., Yan Z., Liao T., Yao W., Wu W., Yu T., Chen Y, & Zhang Y. (2019). Hsa_circ_0058124 promotes papillary thyroid cancer tumorigenesis and invasiveness through the NOTCH3/GATAD2A axis. Journal of Experimental & Clinical Cancer Research : CR, 38, 318.

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Immunohistochemical Analysis of Apoptosis

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Paraffin-embedded tissue sections were sequentially deparaffinized and rehydrated in xylene, 100% ethanol, 90% ethanol, 70% ethanol, and distilled water. Deparaffinized sections were autoclaved in 10 mM sodium citrate buffer (pH 6.0) for antigen retrieval (15 min at 121 °C), and incubated in 3% hydrogen peroxide in methanol (10 min at room temperature) to quench endogenous peroxidase activity. After incubation for 1 h in blocking buffer (10% normal goat serum in PBS), sections were stained with anti-cleaved caspase-3 (Asp175) antibody (1:200 dilution), followed by a peroxidase- and a second antibody-conjugated amino acid polymers (Nichirei Biosciences, Tokyo, Japan). Staining was developed using a DAB Kit (Vector Laboratories). Sections were counterstained with hematoxylin. For counting the number of neointimal cells, deparaffinized sections were stained with hematoxylin and eosin and were subsequently analyzed using the ImageJ 1.48j software (NIH).

Ashino T., Yamamoto M, & Numazawa S. (2016). Nrf2/Keap1 system regulates vascular smooth muscle cell apoptosis for vascular homeostasis: role in neointimal formation after vascular injury. Scientific Reports, 6, 26291.

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Comprehensive Protein Expression Analysis

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Western blot reagents were obtained from GE Healthcare (Pittsburgh, PA, USA). Fluorescent probes for caspase activity, caspase inhibitors, and annexin V assay kit were from BD Biosciences (San José, CA, USA). Culture media were from Lonza (Basel, Switzerland). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and p38MAPK inhibitor (SB202190) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary monoclonal rabbit antibodies against caspase 8 (dilution, 1:1,000; #4927), cleaved-caspase 9 (dilution, 1:1,000; #7237), MMP-2 (dilution, 1:1,000; #4022), MMP-9 (dilution, 1:1,000; #3852), p-p38 (dilution, 1:1,000; #9211), p38 (dilution, 1:1,000; #9212), and TIMP-1 (dilution, 1:1,000; #8946) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Caspase 3 (dilution, 1:1,000; sc-7148), Bid (dilution, 1:1,000; sc-11423), Bcl-2 (dilution, 1:1,000; sc-783), Bcl-xL (dilution, 1:1,000; sc-634), and Bax (dilution, 1:1,000; sc-526) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), and β-actin (dilution, 1:5,000; #A5441) was obtained from Sigma-Aldrich (St. Louis, MO, USA).
Anti-Ki67 (dilution 1:200) and Click-iT Tunel colorimetric IHC detection kit were from Thermo Fisher (Waltham, MA USA). DAB kit was provided from Vector laboratories (Burlingame, CA, USA).

Sánchez-Martín V., Jiménez-García L., Herranz S., Luque A., Acebo P., Amesty Á., Estévez-Braun A., de las Heras B, & Hortelano S. (2019). α-Hispanolol Induces Apoptosis and Suppresses Migration and Invasion of Glioblastoma Cells Likely via Downregulation of MMP-2/9 Expression and p38MAPK Attenuation. Frontiers in Pharmacology, 10, 935.

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Immunohistochemical Analysis of Prostate Lobes

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Prostate lobes were separated by microdissection, and embedded in paraffin for sectioning ^{24 (link)}. After antigen retrieval in hot citrate buffer, sections were blocked in 5% of normal horse serum and 1% of normal goat serum, and subjected to immunohistochemistry staining using the ABC Elite Kit and the DAB Kit (Vector) according to the manufacturers’ recommendations. The following antibodies were used: Ki-67 (ab15580, 1:600) from Abcam; ATF3 (Santa Cruz sc-188, 1:200), AR (sc-816, 1:200), and p63 (sc-8430, 1:200) from Santa Cruz; p-AKT S473 (#4060, 1:200), p-AKT T308 (#2965,1:200), AKT (#4691, 1:200), p-S6 (#2211, 1:400), S6 (#2217, 1:200), cleaved caspase-3 (#9661, 1:300), and Pten (#9188, 1:100)from Cell Signaling; MMP2 (NB200-193, 1:200) from Novus; and MMP-9 (ab137867, 1:1000) from Abcam. For α-smooth muscle actin (α-SMA) staining, sections were incubated with alkaline phosphatase (AP)-conjugated anti-α-smooth muscle actin antibody (Sigma, 1:600) followed by detection of AP activity using the SIGMAFAST Fast Red TR/Naphthol AS-MX tablets (F4523, Sigma) according to the supplier’s protocol. For quantifying IHC staining intensity, random microscopic fields were captured and digitized by a CCD camera (Olympus). Signal intensity was determined using the Image-Pro Plus software and presented as integrated optical density (IOD).

Wang Z., Xu D., Ding H.F., Kim J., Zhang J., Hai T, & Yan C. (2014). Loss of ATF3 Promotes Akt Activation and Prostate Cancer Development in a Pten Knockout Mouse Model. Oncogene, 34(38), 4975-4984.

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Immunohistochemical Analysis of Neuroinflammation

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For immunohistochemistry (IHC), the brains were separated in PFA solution at 4°C overnight and embedded with 3% agarose in PBS. Coronal sections were gathered using VT1000 vibratome (Leica Biosystems, Wetzlar, Germany) at a thickness of 30 μm throughout the brain, including the SNpc and striatum and every 3 sections were analyzed. 3% H₂O₂ was used to block endogenous peroxidase activity for 10 min at room temperature and sections were blocked with 10% donkey serum for 10 min. For immunofluorescence (IF), sections were blocked with 10% donkey serum for 30 min. Then, the sections were incubated with primary antibody. The sections were incubated with an HRP-conjugated anti-rabbit antibody, stained using the DAB kit (Vector Labs, Carlsbad, CA, USA), and were incubated overnight at 4°C. Fluorescein was combined with a fluorescent secondary antibody. Images were gathered using a fluorescence microscope (Olympus, Tokyo, Japan). The quantifications were performed using ImageJ. The following antibodies were used: anti-Iba1 antibody (Cat# ab5076, Abcam), anti-GFAP antibody (G3893, Sigma-Aldrich, St Louis, MO, USA), anti-α-syn antibody (Cat# ab212184, Abcam), anti-pSer129-α-syn antibody (Cat# ab51253, Abcam), Goat anti-rabbit antibody (Cat# ab6721, Abcam), donkey anti-mouse antibody (Cat# A21203, Invitrogen, Carlsbad, CA, USA), donkey anti-goat antibody (Cat# A32758, Invitrogen).

Cui C., Han Y., Li H., Yu H., Zhang B, & Li G. (2022). Curcumin-driven reprogramming of the gut microbiota and metabolome ameliorates motor deficits and neuroinflammation in a mouse model of Parkinson’s disease. Frontiers in Cellular and Infection Microbiology, 12, 887407.

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