Dab solution

Manufactured by Agilent Technologies

Sourced in Denmark, United States, Germany

The DAB solution is a laboratory reagent used in various analytical and diagnostic applications. It serves as a chromogenic substrate for the detection of specific target molecules or enzymes in samples. The solution contains 3,3'-Diaminobenzidine, a chemical compound that produces a brown-colored precipitate when it reacts with the target analyte, allowing for visual identification and localization.

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Close spelling variants of this product

DAB+ chromogen solution Diaminobenzidine (DAB) solution DAB peroxidase substrate solution 3,3′‐diaminobenzidine (DAB) chromogen substrate solution DAB work solution DAB substrate solution 3’-3’-diamobenzidine (DAB) chromogen-substrate solution DAB substrate-chromogen solution Diaminobenzidine substrate-chromogen solution (DAB, 3,3′-diaminobenzidine (DAB) solution

64 protocols using dab solution

Immunohistochemical Analysis of Ki67 Expression

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Sectioned tumour slides were deparaffinized and rehydrated. The sections were
incubated in antigen retrieval (pH 9.0), washed with TBST washing buffer and
incubated with Dako^® peroxidase blocking reagent for 1 h. The
sections were then washed with TBST (three times, 5 min per wash). The slides
were blocked using blocking solution (10% goat serum and 5% BSA in TBST) for
1 h. Slides were then washed again and incubated with primary antibodies against
Ki67 (Dako, Clone MIB-1, Cat#M7240, mouse monoclonal) overnight at 4°C.
Subsequently, the slides were washed and incubated with biotinylated secondary
antibody for 1 h, followed by washing with TBST (three times, 5 min each) to
remove unbounded antibodies. The sections were incubated with ABC solution for
1 h, and remaining solutions were removed through washing with TBST as
previously described. Finally, 200 µl of Dako^® DAB solution was
applied to each section, and samples were monitored closely to examine the
development of stains. The slides were counterstained with haematoxylin, washed,
left to dry overnight and mounted. Slides were imaged using a light microscope
(CX41, Olympus) at 100× magnification. The ratio of stained cells
versus total cells were scored using Image J software.

Tan Y.J., Lee Y.T., Petersen S.H., Kaur G., Kono K., Tan S.C., Majid A.M, & Oon C.E. (2019). BZD9L1 sirtuin inhibitor as a potential adjuvant for sensitization of colorectal cancer cells to 5-fluorouracil. Therapeutic Advances in Medical Oncology, 11, 1758835919878977.

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Immunohistochemical Analysis of VEGFR-3 and CXCR4

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The expression of VEGFR-3 and CXCR4 was analyzed by immunohistochemistry (IHC). Paraffin-embedded tissue samples were obtained from 70 patients for CXCR4 and 69 for VEGFR-3, due to limited availability of material. The nature of the collected material was mainly tumour resections.
Three micrometer thick tissue sections were cut and mounted on super frost slides. These were deparaffinized, rehydrated and peroxidase blocked (3% H₂O₂ in methanol, 30 min). After blocking of nonspecific protein binding sites by using fresh frozen plasma (30 min) slides were incubated with the respective primary antibody VEGFR-3 (sc 321, Santa Cruz Biotechnology, UK, 1:200, 2 h) and CXCR4 (CIO115, Capralogics, USA, 1:300, 1.5 h) at room temperature, as described before [30 (link)-32 (link)]. Incubation with secondary antibody (anti-rabbit–mouse–goat antibody) was followed by incubation with streptavidin–POD (DAKO, Germany, each 15 min). Specific antibody binding was visualized using DAB solution (DAKO, Germany) and the tissues were counterstained by hemalaun solution (DAKO, Germany). Between each step of staining the specimens were washed in distilled water or DPBS.
Evaluation of staining was performed by two independent, blinded pathologists.

Thomaidis T., Maderer A., Al-Batran S.E., Kany J., Pauligk C., Steinmetz K., Schad A., Hofheinz R., Schmalenberg H., Homann N., Galle P.R, & Moehler M. (2014). VEGFR-3 and CXCR4 as predictive markers for treatment with fluorouracil, leucovorin plus either oxaliplatin or cisplatin in patients with advanced esophagogastric cancer: a comparative study of the Arbeitsgemeinschaft Internistische Onkologie (AIO). BMC Cancer, 14, 476.

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Immunohistochemical Analysis of Smad3

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The fresh specimens were fixed in 4% formalin and embedded in paraffin blocks, sectioned into slices, dewaxed and rehydrated. After antigen retrieval and blocking with bovine serum albumin, the tissues were incubated overnight at 4°C with anti-Smad3 (Proteintech, US). Incubation for 50 mins at room temperature with the according specific secondary antibody (HRP, Dako, Denmark), and staining with the DAB solution (Dako, Denmark), which stood for brown color development, followed. The intensity and proportion of cells were both assessed for semi-quantification of the strength of positivity.

Pang Q., Wang Y., Xu M., Xu J., Xu S., Shen Y., Xu J, & Lei R. (2019). MicroRNA-152-5p inhibits proliferation and migration and promotes apoptosis by regulating expression of Smad3 in human keloid fibroblasts. BMB Reports, 52(3), 202-207.

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Immunohistochemical Analysis of CXCR4 in TMAs

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Immunohistochemical staining was performed on tissue microarray (TMA). Tissue microarrays were established as previously described (Zhu et al, 2008 (link)). Primary antibody against human CXCR4 (1 : 400 dilution, R&D systems, Minneapolis, MN, USA) was applied in the procedure. The sections were heated at 70 °C for 1 h, dewaxed in xylene, and dehydrated through a gradient concentration of alcohol. After retrieving and blocking the endogenous peroxidase and non-specific staining with 3% (v/v) HR2ROR2R and normal goat serum, the sections were incubated with anti-CXCR4 antibody overnight at 4 °C. The slides were then incubated with HRP-conjugated goat anti-mouse IgG secondary antibody for 10 min at 37 °C. Finally, the sections were visualised by DAB solution (DAKO, Carpinteria, CA, USA) and counterstained with haematoxylin (DAKO). Staining intensities and percentages of positive tumour cells were scored independently by two pathologists who were blind to the patients' outcome. A five-staged score (0, 1, 2, 3, and 4) was deducted from these two parameters according to a previously described scheme (Went et al, 2005 (link)).

An H., Xu L., Zhu Y., Lv T., Liu W., Liu Y., Liu H., Chen L., Xu J, & Lin Z. (2014). High CXC chemokine receptor 4 expression is an adverse prognostic factor in patients with clear-cell renal cell carcinoma. British Journal of Cancer, 110(9), 2261-2268.

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Immunohistochemical Analysis of CSNK2A1 Protein

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CSNK2A1 protein expression was determined via immunohistochemistry (IHC) analysis using paraffin-embedded liver sections. Anti-CSNK2A1 IHC staining was carried out using the LsAB Kit (DAKO, Glostrup, Denmark). All tissue sections were de-waxed, treated with Proteinase K enzyme, and the endogenous peroxidase activity was blocked by incubating with 3% hydrogen peroxide for 10 min. After washing with phosphate-buffered saline (PBS; pH 7.6) for 5 min, the slides were incubated with anti-CSNK2A1 antibodies (GTX84369; GeneTex, Hsinchu, Taiwan) for 30 min at 37 ℃, followed by rabbit anti-rat antibodies and a goat anti-rabbit HRP polymer for 15 min. The immunocomplexes were visualized using DAB solution (DAKO) for 5 min. Samples were washed with PBS (pH 7.6) in order to perform all the necessary steps 14 (link),15 (link),16 .

Lan Y.C., Wang Y.H., Chen H.H., Lo S.F., Chen S.Y, & Tsai F.J. (2020). Effects of Casein Kinase 2 Alpha 1 Gene Expression on Mice Liver Susceptible to Type 2 Diabetes Mellitus and Obesity. International Journal of Medical Sciences, 17(1), 13-20.

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Quantifying MTH1 Expression in Tissue

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The disks from humans and mice were fixed in 4% paraformaldehyde for one week, decalcified in 20% EDTA for two weeks, paraffin-embedded, and then carefully sectioned to a 7 μm thickness. After deparaffinization, antigen retrieval, and blocking with 5% goat serum, the slides were incubated with primary antibody: MTH1 (diluted 1:200, Cat. #ab197028; Abcam) and secondary antibody (DAKO). Subsequently, sections were developed with DAB solution (DAKO, Denmark) and then counterstained with hematoxylin. Histological images were acquired using an Olympus BX63 microscope (Olympus) with randomly selected fields in each section at ×400 magnification. The percentages of MTH1⁺ cells were quantified using ImageJ software (National Institutes of Health). We considered <50% positive staining as low expression of MTH1 and ≥50% positive staining as high expression of MTH1.

Zhang J., Liu R., Mo L., Liu C, & Jiang J. (2022). miR-4478 Accelerates Nucleus Pulposus Cells Apoptosis Induced by Oxidative Stress by Targeting MTH1. Spine, 48(5), E54-E69.

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Insulin and Glucagon Immunohistochemistry

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Antigen retrieval was performed on paraffin-embedded sections with Retrievagen A Solution (BD Biosciences), and endogenous biotin was blocked by Dual Endogenous Enzyme Blocking Reagent (Dako). Guinea pig antibody to insulin (Dako) or rabbit antibody to glucagon (Abcam) was added and detected with the EnVision Dual Link Kit (Dako) followed by staining with DAB Solution (Dako). Samples were counterstained with hematoxylin. A veterinary pathologist scored histopathological changes by blinded scoring of sections.

Gallagher G.R., Brehm M.A., Finberg R.W., Barton B.A., Shultz L.D., Greiner D.L., Bortell R, & Wang J.P. (2014). Viral Infection of Engrafted Human Islets Leads to Diabetes. Diabetes, 64(4), 1358-1369.

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Quantifying Tumor Angiogenesis via CD31 Staining

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CD31 in tissue sections was stained using a rat anti-mouse CD31 monoclonal antibody (BD Biosciences, San Jose, CA, USA) as described previously with some modifications^{45 (link)}. In brief, rat anti-mouse CD31 monoclonal antibody bound to CD31 in tissue sections was detected by using Rat-HRP Polymer, 1-Step (Biocare Medical, Concord, CA, USA) containing XM Factor (Biocare Medical) followed by using DAB solution (Dako, Carpinteria, CA, USA).
Viable tumour areas were determined for each tumour tissue section by using a fluorescence microscope (BZ-X710; Keyence Corporation, Osaka, Japan). The CD31-stained area and the total viable cell area were calculated by using BZ-Analyzer (version 1.2.0.1; Keyence Corporation). Microvessel density was calculated by dividing the CD31-stained area by the total viable cell area.

Nishidate M., Yamamoto K., Masuda C., Aikawa H., Hayashi M., Kawanishi T, & Hamada A. (2017). MALDI mass spectrometry imaging of erlotinib administered in combination with bevacizumab in xenograft mice bearing B901L, EGFR-mutated NSCLC cells. Scientific Reports, 7, 16763.

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Immunohistochemistry of Thymus Apoptosis

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Thymus was fixed in fresh 4% paraformaldehyde for 24 hr and stored in 70% ethanol until paraffin embedding. Hematoxylin and eosin staining were performed on 5 μm–thick paraffin sections. For cleaved caspase-3 IHC, the anti-cleaved caspase-3 antibody (1:50, Cell Signaling Technology #9661) was used. Slides were incubated with Envision Labeled Polymer-HRP Anti-Rabbit (Dako, K4002) for 30 min. Sections were developed with DAB+ solution (Dako, K3468) and counterstained with Harris Hematoxylin. Imaging analysis was performed with ImageJ (FIJI) to automatically count the labeled cells in each region, the data were shown as number of positive cells per region.

Liu X., Yu J., Xu L., Umphred-Wilson K., Peng F., Ding Y., Barton B.M., Lv X., Zhao M.Y., Sun S., Hong Y., Qi L., Adoro S, & Chen X. (2021). Notch-induced endoplasmic reticulum-associated degradation governs mouse thymocyte β−selection. eLife, 10, e69975.

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Immunohistochemical Analysis of Tissue Samples

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Tissue specimens were fixed in neutral formalin buffered saline (10%)
and embedded in paraffin. Hematoxylin and eosin staining was performed using
standard methods and tissue specimens from experimental animals were reviewed in
a blinded fashion by a clinical pathologist (M. M. I.) For Ki67 staining,
3–4 micron tissue sections were cut from paraffin blocks and baked
overnight in a dry slide incubator, then deparaffinized on a Shandon-Lipshaw
Varistain using a series of incubations in xylene, ethanol, then water. Antigen
retrieval was achieved by incubating slides in Tris-HCL 9.0 AR buffer in a T-FAL
OPTIMA pressure cooker. Slides were rinsed, then endogenous peroxidase was
blocked by immersing slides in 3% hydrogen peroxide for 5 minutes. Following
washes in nanopure water and TBS-20, primary antibody was applied at a dilution
of 1:200 (Ki67, MIB-1 Clone, Dako). Following primary antibody incubation,
slides were washed and then incubated with envision-labelled polymer-HRP
Anti-Mouse (Dako). Slides were washed, then DAB+ solution (DakoCytomation) was
added for a 15-minute incubation, after which slides were rinsed with nanopure
water. Chromogen signal was enhanced using DAB Sparkle Enhancer (Biocare).
Slides were washed, then counterstained with Harris Hematoxylin, dehydrated,
cleared, then mounted using Cytoseal (VWR).

Bader D.A., Hartig S.M., Putluri V., Foley C., Hamilton M.P., Smith E.A., Saha P.K., Panigrahi A., Walker C., Zong L., Martini-Stoica H., Chen R., Rajapakshe K., Coarfa C., Sreekumar A., Mitsiades N., Bankson J.A., Ittmann M.M., O’Malley B.W., Putluri N, & McGuire S.E. (2018). Mitochondrial pyruvate import is a metabolic vulnerability in androgen receptor-driven prostate cancer. Nature metabolism, 1(1), 70-85.

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