Vector dab substrate kit

Manufactured by Vector Laboratories

Sourced in United States

The Vector DAB substrate kit is a laboratory product that provides a chromogenic substrate for the visualization of target proteins in immunohistochemistry and related applications. The kit includes a diaminobenzidine (DAB) solution and other necessary reagents to facilitate the detection and localization of specific antigens within biological samples.

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Lab products found in correlation

Hematoxylin, by Vector Laboratories (2 mentions) Anti phospho h3antibody, by Cell Signaling Technology (1 mentions) Vector vip substrate kit, by Vector Laboratories (1 mentions) Histoclear, by Merck Group (1 mentions) Impress reagent anti rabbit igg, by Vector Laboratories (1 mentions) Permount, by Thermo Fisher Scientific (1 mentions) Stereo investigator software, by MBF Biosciences (1 mentions) Orca r2 digital camera, by Hamamatsu Photonics (1 mentions) Axioskop 2 microscope, by Zeiss (1 mentions) Vectastain abc kit, by Vector Laboratories (1 mentions) Fl 393, by Santa Cruz Biotechnology (1 mentions) Rabbit anti ki67, by Abcam (1 mentions) Vectastatin elite abc kit, by Vector Laboratories (1 mentions) Anti ki67 antibody, by Abcam (1 mentions) Anti e cadherin, by Cell Signaling Technology (1 mentions) Anti vimentin, by Cell Signaling Technology (1 mentions) Trypsin, by Merck Group (1 mentions)

Close spelling variants of this product

Vector DAB substrate DAB substrate kit Vector DAB kit DAB substrate Vector® DAB DAB kit Substrate kits

14 protocols using vector dab substrate kit

Immunohistochemical Analysis of GC Tissues

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GC tissue microarray sections (4 µm) were heated at 60 °C for 2 h. After dewaxing, rehydration and antigen retrieval, tissue sections were blocked with 5% BSA and incubated with corresponding primary antibodies overnight at 4 °C then biotinylated secondary antibody for 30 min at 37 °C. Vector DAB Substrate Kit (Vector Laboratories, CA) was used to control the degree of chromogenic reaction. Tissue sections were then counterstained with hematoxylin and colored with lithium carbonate. Stained sections were scanned using a Pannoramic 250 FLASH Slide scanner. Antibodies used are indicated in Table S8, Supporting Information.

Wu Z., Qu B., Yuan M., Liu J., Zhou C., Sun M., Guo Z., Zhang Y., Song Y, & Wang Z. (2023). CRIP1 Reshapes the Gastric Cancer Microenvironment to Facilitate Development of Lymphatic Metastasis. Advanced Science, 10(26), 2303246.

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Immunostaining Zebrafish Embryos

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16a and 16e were microinjected into one-cell stage zebrafish embryos using a PV820 Pneumatic PicoPump (WPI, Sarasota, FL, USA). For immunostaining, embryos were fixed in 4% paraformaldehyde. Embryos were rinsed three times in phosphate-buffered saline (PBS), in cold methanol for 5 min, and then rinsed in MeOH/PBS-Tween 20 (PBST) and PBST for each 5 min. Embryos were blocked for 3 h using Blocking Reagent (5% BSA/ 10% goat serum in PBS/1% DMSO/0.5% Triton-X100/0.1% Tween 20) with mild rotating. Embryos were incubated overnight at 4 °C with a polyclonal anti-phospho-H3 antibody (1:1000, Cell Signaling Technology, Danvers, MA, USA). Embryos were washed 4× in PBDTT (1 h each) before addition of biotin-labeled goat anti-rabbit antibody (1:2000) in 1% BSA/PBDTT after blocking for 4 h. Antibody staining was detected using the Vectastain Elite ABC reagent and Vector DAB substrate kit to the manufacturer’s specifications. Embryos used in this study were obtained from the Zebrafish Center for Disease Modeling (ZCDM; Daejeon, Republic of Korea). Animal experiments were conducted according to guidelines approved by the Animal Care and Use Committee of Chungnam National University.

Ryu S., Park J.E., Ham Y.J., Lim D.C., Kwiatkowski N.P., Kim D.H., Bhunia D., Kim N.D., Yaffe M.B., Son W., Kim N., Choi T.I., Swain P., Kim C.H., Lee J.Y., Gray N.S., Lee K.S, & Sim T. (2022). Novel Macrocyclic Peptidomimetics Targeting the Polo-Box Domain of Polo-Like Kinase 1. Journal of medicinal chemistry, 65(3), 1915-1932.

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Immunohistochemistry Protocol for Protein Analysis

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Immunohistochemistry was performed as previously described (12 (link)). Antibodies were used at the following dilutions: anti-phospho(Ser473)Akt (1:25), anti-PTEN (1:100), anti-NUAK2 (1:100), anti-p27 (1:100) and anti-CDK2 (1:2000). For immunostaining of CDK2 and p27, staining was developed with a Vector VIP Substrate Kit (Vector Laboratories, Burlingame, CA) in pigmented melanomas or with a Vector DAB Substrate Kit (Vector Laboratories) using counter-staining in non-pigmented melanomas. Cells stained in the nucleus and/or both the nucleus and cytoplasm were counted as positive, and cells that stained only in the cytoplasm were counted as negative. Immunostaining of CDK2 was scored from 0 to +3 (0 = 0 to10%, +1 =11% to 25%, +2 = 26% to 50% or +3 = 51% to 100%) depending on the percentages of cells in a blind fashion by 3 observers. The basal expression group (negative staining group) includes specimens with a 0 score and the over-expression group (positive staining group) includes specimens with +1, +2 or +3 scores.
Immunohistochemistry using fluorescence was performed as previously described (12 (link)) using the anti-p21 antibody (1:100). Images were captured using a Leica DMR B/D MLD fluorescence microscope (Leica, Weltzar, Germany) and a Dage-MTI 3CCD 3-chip color video camera (Dage-MTI, Michigan City, IN).

Namiki T., Yaguchi T., Nakamura K., Valencia J.C., Coelho S.G., Yin L., Kawaguchi M., Vieira W.D., Kaneko Y., Tanemura A., Katayama I., Yokozeki H., Kawakami Y, & Hearing V.J. (2015). NUAK2 amplification coupled with PTEN deficiency promote melanoma development via CDK activation. Cancer research, 75(13), 2708-2715.

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Immunohistochemistry of Placental Tissue

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Immunohistochemistry procedure was performed as previously described^{5 (link)}. Briefly, paraffin embedded human placental tissue samples were sectioned, deparaffinized, and hydrated with Histoclear and ethanol (Sigma, St. Louis, MO, USA). Slides then underwent antigen retrieval using 10 mM sodium citrate (pH 6) buffer in a pressure cooker. Sections were blocked using DAKO Dual Endogenous Enzyme Block for Autostainer and 2.5% normal horse serum (Agilent, Santa Clara, CA, USA). Slides were then incubated with primary antibody m28-RNLS (1:500) overnight at 4 °C and secondary antibody IMPRESS Reagent anti-Rabbit IgG (Vector Laboratories, Burlingame, CA, USA). Antibody binding was detected using Vector DAB substrate kit and counterstained with hematoxylin (Vector Laboratories, Burlingame, CA, USA). Images were obtained at 20× and 60× using Echo^® Revolve microscope. Staining specificity was determined by labeling with secondary antibody.

Wang M., Silva T., Toothaker J.M., McCourt B.T., Shugrue C., Desir G., Gorelick F, & Konnikova L. (2022). Renalase and its receptor, PMCA4b, are expressed in the placenta throughout the human gestation. Scientific Reports, 12, 4953.

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Pimonidazole Hypoxia Detection Protocol

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Mice were injected intraperitoneally with 60mg/kg pimonidazole solution (Hypoxyprobe™-1, Hypoxyprobe Inc., Burlington, MA, USA). One hour later, tumors were excised, paraffin embedded and cut into 5μm sections. Tissue sections were de-waxed, rehydrated and incubated in 3% H₂O₂ to quench endogenous peroxidase. After washing with PBS, antigen was retrieved by boiling in 10mM citrate buffer (pH 7.0) for 20min. Sections were cooled to RT, washed and blocked with 1% BSA for 1h at RT. Sections were incubated with anti-pimonidazole monoclonal antibody (MAb1), at a dilution of 1:50 in 1% BSA for 1h at RT. After washing, sections were incubated with the HRP-conjugated secondary antibody for 1h at RT. Reactivity was visualized using Vector DAB substrate kit (Vector laboratories Inc.). The sections were counterstained with Meyer’s hematoxylin for 2min, washed, dehydrated and mounted with Permount (Thermo Fisher Scientific). Sections were visualized under light microscope and photographed.

Ryan D., Bogan D., Davies J., Koziol J, & ElShamy W.M. (2017). A niche that triggers aggressiveness within BRCA1-IRIS overexpressing triple negative tumors is supported by reciprocal interactions with the microenvironment. Oncotarget, 8(61), 103182-103206.

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Immunohistochemical Analysis of Amyloid-beta

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Periventricular brain tissue (100 μm thick, as above) was subjected to antigen retrieval consisting of boiling in 10 mM sodium citrate, pH 6, for 10 min followed by three PBS washes, then treatment in 88% formic acid (ThermoFisher Scientific) for 10 min. Tissue was then washed three times in PBS and endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide for 10 min. After three PBS washes, tissue was blocked for 1 h at RT in 10% (v/v) milk in PBS followed by incubation for 24 h at 4°C with anti-Aβ42 clone D9A3A 14974 (1:1000; Cell Signaling Technology) in blocking solution. Whole mounts and sections were then washed three times for 10 min each with 0.1% Triton X-100 in PBS and incubated for 1 h at RT with biotinylated anti-rabbit secondary BA-1000, 1:300 (Vector Laboratories). Biotinylated secondary antibody detection was completed using the Vectastain ABC Kit and visualized using the Vector DAB Substrate Kit (Vector Laboratories) according to the manufacturer’s instructions. The sections were imaged using Stereo Investigator software (MBF Bioscience) with a Zeiss Axioskop 2+ microscope (Carl Zeiss MicroImaging) and an Orca-R2 digital camera (Hamamatsu Photonics).

Todd K.L., Brighton T., Norton E.S., Schick S., Elkins W., Pletnikova O., Fortinsky R.H., Troncoso J.C., Molfese P.J., Resnick S.M, & Conover J.C. (2018). Ventricular and Periventricular Anomalies in the Aging and Cognitively Impaired Brain. Frontiers in Aging Neuroscience, 9, 445.

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Immunohistochemical Detection of Mdm4 and p53

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Mdm4 immunohistochemistry (IHC) was performed as described previously using AB112 antibody [13 (link)]. FL-393 (Santa Cruz Biotechnology) was used for p53 IHC (1:50 dilution). Vector DAB Substrate Kit (Burlingame, CA) was used for chromogenic detection.

Xiong S., Pant V., Zhang Y., Aryal N.K., You M.J., Kusewitt D, & Lozano G. (2017). The p53 inhibitor Mdm4 cooperates with multiple genetic lesions in tumorigenesis. The Journal of pathology, 241(4), 501-510.

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Immunohistochemical Analysis of Cell Markers

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For IHC, sections were rehydrated followed by heat-induced epitope retrieval and incubated for 10 min with 0.3% H₂O₂ in water to block endogenous peroxidase. Sections were incubated with blocking buffer (10% normal goat serum diluted in PBS) for 60 min at room temperature and then with a primary antibody diluted in blocking buffer overnight at 4 °C. Primary monoclonal antibodies used were E-CADHERIN, γ-CATENIN, VIMENTIN and FIBRONECTIN-1 (Supplementary Table 6). The following day, the biotinylated secondary antibody was used to incubate the sections for 60 min at room temperature. And then, streptavidin/biotin HRP-conjugate was added to the sections for 30 min. The signal was developed using the Vector DAB substrate kit according to the manufacturers’ instructions.

Liu Y., Chen C., Qian P., Lu X., Sun B., Zhang X., Wang L., Gao X., Li H., Chen Z., Tang J., Zhang W., Dong J., Bai R., Lobie P.E., Wu Q., Liu S., Zhang H., Zhao F., Wicha M.S., Zhu T, & Zhao Y. (2015). Gd-metallofullerenol nanomaterial as non-toxic breast cancer stem cell-specific inhibitor. Nature Communications, 6, 5988.

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Quantifying Pancreatic Cell Proliferation

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Pancreatic tissue proliferation in response to injury was monitored by immunostaining of the nuclear proliferation marker Ki67. Pancreatic paraffin tissue sections were exposed to antigen-retrieval in boiling Tris-EDTA Buffer (10 mM Tris Base, 1 mM EDTA Solution, 0.05% Tween 20, pH 9.0), followed by antibody incubation and detection using the ABC-Vectastain system with the Vector DAB substrate kit (Vector Laboratories, INC, Burlingame, CA). Sections were counterstained with Eosin. Primary antibody was rabbit anti-Ki67 (Abcam, Cambridge, UK). Quantification was performed by counting Ki67 positive cells in an entire tissue section using a 20× objective. The image of the whole tissue section was captured with a digital camera (Canon, PowerShot, ELPH 300HS). The total area (mm²) of the entire tissue sections was measured with the Image J program (v1.46r; NIH), and the number of Ki67 positive cells per mm² area was determined.

Zhang L., Kline RH I.V., McNearney T.A., Johnson M.P, & Westlund K.N. (2014). Cannabinoid receptor 2 agonist attenuates pain related behavior in rats with chronic alcohol/high fat diet induced pancreatitis. Molecular Pain, 10, 66.

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Xenograft Study of Anti-Tumor Compounds

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Six-week-age female Balb/c nude mice were subcutaneously injected into the abdominal region with HT1080 cells (2 × 10⁶ cell/200 µl PBS). On day 7 post-inoculation of tumor cell when tumor volume reached to about 50–100 mm³, mice were randomly divided into 6 groups (n = 6 per group) and daily administered with saline (control), SR, VF, SRVF, and SR + VF in a volume of 100 µl for 14 days. During experiments, mice were carefully observed for the appearance, motility, and behavior. In addition, tumor volume was measured twice per week. On day 21, tumors were excised and weighed after euthanasia by inhalation of isoflurane. To assess the safety of SR, VF, and SRVF, 6-week-old female Balb/c nude mice were divided into the 5 groups (n = 3 per group) and fed with vehicle (saline), SR, VF, or SRVF daily during 14 days. At the time of sacrifice, weights of major organs and body weight were measured. For the detection of proliferating cells in xenograft tumors, immunochemical staining for Ki67 was performed on 20 µm-thick cryosections using anti-Ki67 antibody (Abcam, Cambridge, MA, USA) and avidin-biotinylated enzyme complex (ABC) with DAB staining (Vectastatin Elite ABC kit, Vector DAB Substrate kit, Vector Laboratories, Burlingame, CA, USA).

Kim A., Im M, & Ma J.Y. (2017). SRVF, a novel herbal formula including Scrophulariae Radix and Viticis Fructus, disrupts focal adhesion and causes detachment-induced apoptosis in malignant cancer cells. Scientific Reports, 7, 12756.

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