Vectastain avidin biotin complex kit

Manufactured by Vector Laboratories

Sourced in United States

The Vectastain Avidin-Biotin Complex (ABC) kit is a tool used in immunohistochemistry and related techniques. It provides a method for amplifying the signal of a target antigen detected by a biotinylated primary antibody. The kit contains the necessary components to form an avidin-biotin complex that can be detected, thereby enhancing the visualization of the target antigen.

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Lab products found in correlation

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Spelling variants of this product

Avidin-biotin complex (175 citations) Vectastain kit (77 citations)

15 protocols using vectastain avidin biotin complex kit

Endometrial GRP78 Expression Evaluation

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Archival endometrial samples were evaluated for GRP78 as previously described [11 (link),12 (link)]. Briefly, freshly cut formalin-fixed, paraffin-embedded samples were deparaffinized and rehydrated. Antigen retrieval was accomplished with BD Retrievagen A (BD Pharmingen, Carpenteria, CA) according to manufacturer instructions. GRP78 was detected with rabbit anti-GRP78 (1:100, Santa Cruz Biotechnologies, Santa Cruz, CA) followed by the goat anti-rabbit antibody (VECTASTAIN® avidin-biotin complex (ABC) kit, Vector Laboratories, Burlingame, CA). Visualization was achieved with 3,3′-diaminobenzidine (DAB).
The study pathologist (PMF), blinded to clinical and treatment data, scored all specimens as negative, weak, moderate and strong. Negative controls absent of primary antibody were used as a reference. Overexpression of GRP78 was defined as moderate/strong intensity [12 (link)]. Internal consistency was evaluated by re-presenting a random subset of previously scored slides to the same pathologist, blinded to the original score.

Tierney K.E., Ji L., Dralla S.S., Yoo E., Yessaian A., Pham H.Q., Roman L., Sposto R., Mhawech-Fauceglia P, & Lin Y.G. (2016). Endoplasmic reticulum stress in complex atypical hyperplasia as a possible predictor of occult carcinoma and progestin response. Gynecologic oncology, 143(3), 650-654.

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Quantifying Vascular Smooth Muscle Cells Post-Stenting

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At 6 weeks post stent implantation, rabbit aortas were harvested, the stent struts removed and paraffin embedded. They were then deparaffinized, serially washed, incubated in 3% H2O2, washed, blocked for 10 min with 10% goat serum, followed by the respective primary antibody diluted in 1:50 blocking solution and incubated at 4C overnight. Primary antibodies included: SMC (monoclonal, actin antibody, mouse anti-rabbit, MA5-11869, Invitrogen, Carlsbad, CA) and ADORA-2B (polyclonal, goat anti-Human, Novus Biological, Oakville, ON, Canada). Following washing, Vectastain Avidin-Biotin Complex (ABC) kit (Vector Laboratories, PK-6100) was utilized per manufacturer's instruction s (Vector Laboratories, Newark, CA, USA). Biotinylated secondary antibody was then incubated for 10 min at room temperature and washed. Detection was then performed with DAB (3,3′-Diaminobenzidine) solution (Sigma, D5905) and following preparation, 3% H2O2 was added to the solution. DAB solution was then added to the tissue, incubated and washed. Counterstaining with hematoxylin was then performed for 1 min, washed, incubated in PBS for 2 min and then dehydrated. Slides were then mounted with permount solution for examination. Automated quantification of actin antibody signal intensity within the NI tissue was performed and pixels quantified to compare SMC content in control and DP treated cohorts.

Simard T., Jung R., Di Santo P., Labinaz A., Short S., Motazedian P., Dhaliwal S., Sarma D., Rasheed A., Ramirez F.D., Froeschl M., Labinaz M., Holmes D.R., Alkhouli M, & Hibbert B. (2023). Dipyridamole and vascular healing following stent implantation. Frontiers in Cardiovascular Medicine, 10, 1130304.

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Collagen III Immunohistochemistry in Wound Sections

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A subset of wound plugs was dehydrated, embedded in paraffin, and sectioned at 5 μm. Sections from the middle of the wound were mounted on slides, deparaffinized, stained with a rabbit polyclonal antibody specific for collagen III (Abcam) (2–5 μg/mL), and incubated with a biotinylated polyclonal goat antibody to rabbit IgG (Amersham, Pittsburgh, PA) (5 μg/mL). Collagen III immunostaining was visualized with a Vectastain avidin-biotin complex (ABC) kit (Vector Laboratories, Burlingame, CA) in conjunction with 3,3′-diaminobenzidine.

Damodarasamy M., Johnson R.S., Bentov I., MacCoss M.J., Vernon R.B, & Reed M.J. (2014). Hyaluronan enhances wound repair and increases collagen III in aged dermal wounds. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 22(4), 521-526.

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Immunohistochemical Labeling of Cortical Neurons

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Coronal sections 40 μm-thick containing the sensorimotor cortex were treated with 0.3% H₂O₂ in PBS to block the endogenous peroxidase activity, followed by treating with blocking solution to reduce the non-specific bindings. The sections were then incubated with primary antibodies against NeuN (1:500; Merck Millipore, Darmstadt, Germany), Foxp1 (1:1,000; Abcam, Cambridge, UK), Cux1 (1:1,000; Santa Cruz Biotechnology, Dallas, TX, USA), GAD65/67 (1:2,000; Sigma, St. Louis, MO, USA), Parvalbumin (Pvalb or PV, 1:3,000; Sigma) and Calretinin (Calb2 or CR, 1:3,000; Merck Millipore) at room temperature overnight. Next, sections were incubated with biotinylated secondary antibodies against mouse or rabbit IgG (1:500; The Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 2 h at room temperature followed by Vectastain [avidin-biotin complex (ABC) kit, Vector Laboratories, Burlingame, CA, USA] incubation for 1 h. Last, sections were processed with 2 mg/ml of 3,3′-Diaminobenzidine (DAB) with 0.01% H₂O₂ in PBS and mounted with a glycerol-based aqueous mounting medium.

Lee K.Y., Chang H.C., Seah C, & Lee L.J. (2019). Deprivation of Muscleblind-Like Proteins Causes Deficits in Cortical Neuron Distribution and Morphological Changes in Dendritic Spines and Postsynaptic Densities. Frontiers in Neuroanatomy, 13, 75.

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Immunohistochemical Analysis of Muscle Fiber Composition

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Tissue sections were incubated with diluted mouse anti-myosin fast skeletal antibody (MY-32, 1:2,000; Novus Biologicals, Littleton, CO, USA) or rabbit anti-GLUT4 antibody (1:1,000; Novus Biologicals) overnight at 4℃. After washing, the slides were subsequently incubated with biotinylated goat anti-mouse immunoglobulin G (1:200; Vector Lavoratories, Burlingame, CA, USA) or fluorescein isothiocyanate (FITC)-conjugated antibody (1:50; Novus Biologicals) for 1 hour at room temperature under a foil cover to prevent fluorophore fading. The Vectastain avidin-biotin complex (ABC) kit (Vector Laboratories) was used for the ABC method according to the manufacturer's instructions. Peroxidase activity was visualized with 3,3'-diaminobenzidine tetrachloride (Sigma-Aldrich Co., St. Louis, MO, USA) in 0.1 M PBS. The sections were dehydrated and mounted in Canada balsam (Kanto, Tokyo, Japan). For immunofluorescence, the sections were mounted in Vectashield antifading medium (Vector Laboratories). The images were captured using an inverted microscope (ECLIPSE E100; Nikon, Kanagawa, Japan), or the slides were viewed by fluorescence microscopy (BX50; Olympus, Milan, Italy). The images were analyzed using INFINITYlite software (Innerview 2.0; Lumenera, Ottawa, ON, Canada). Approximately 800 fibers were analyzed in each muscle cross-section.

Kim J.Y., Choi M.J., So B., Kim H.J., Seong J.K, & Song W. (2015). The Preventive Effects of 8 Weeks of Resistance Training on Glucose Tolerance and Muscle Fiber Type Composition in Zucker Rats. Diabetes & Metabolism Journal, 39(5), 424-433.

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Immunohistochemistry of Paraffin Sections

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Tissue sections were deparaffinized and hydrated using decreasing concentrations of ethanol. Antigen retrieval was performed by boiling the slides in 0.01 M trisodium citrate buffer (pH 6.0). Endogenous peroxidases were inactivated by incubating sections in 0.3% hydrogen peroxide for 20 min. To prevent nonspecific binding, sections were pre-incubated with Odyssey buffer for 1 h at room temperature prior to overnight incubation with primary antibody. Biotinylated secondary antibodies were applied for 2 h, and immunoreactivity detected using the Vectastain Avidin-Biotin Complex (ABC) kit (Vector Laboratories) with a 3,3′-diaminobenzidine tetrachloride (DAB) peroxidase substrate kit (Vector Laboratories). All slides were imaged using an upright microscope (Axioskop2; Zeiss MicroImaging Inc.).

McKenzie B.A., Fernandes J.P., Doan M.A., Schmitt L.M., Branton W.G, & Power C. (2020). Activation of the executioner caspases-3 and -7 promotes microglial pyroptosis in models of multiple sclerosis. Journal of Neuroinflammation, 17, 253.

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Quantifying Microglia Activation in Mouse Brains

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Control mice or mice treated with the LPS ± indomethacin regimen described above were euthanized, and their brains removed and fixed in 10 % neutral-buffered formalin, paraffin-embedded, and sectioned at 5 μm. Sections were de-paraffinized, blocked in 2 % goat serum, and exposed to 2–5 μg/ml of mouse anti-F4/80 (Serotec) or rabbit anti-Iba1 (Wako). Sections were then rinsed in Tris-buffered saline and exposed to 5 μg/ml of biotinylated anti-mouse or anti-rabbit IgG and then to Vectastain® avidin-biotin complex (ABC) kit (Vector) in conjunction with 3,3′-diaminobenzidine (DAB, Vector) to derive an insoluble stain that could be quantified.
For quantification, a minimum of two digital images per mouse cerebral cortex were taken at ×20 magnification. The images were processed in ImageJ, and the brightness and contrast adjusted before converting to an RGB stack image. The threshold of the blue stack image is adjusted until the stained tissue shows optimal contrast with highlighting of the DAB stain in red. The staining area and density were measured, and the result was expressed as an area fraction, which is the percent of the enclosed area that is highlighted in red. Relative staining intensity was then calculated for the cerebral cortices for each group of mice.

Banks W.A., Gray A.M., Erickson M.A., Salameh T.S., Damodarasamy M., Sheibani N., Meabon J.S., Wing E.E., Morofuji Y., Cook D.G, & Reed M.J. (2015). Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit. Journal of Neuroinflammation, 12, 223.

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Immunohistochemical Analysis of MMP1 in Lung Tumors

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Formalin-fixed, paraffin-embedded sections were de-waxed, hydrated through decreasing grades of alcohol, and treated with 0.6% hydrogen peroxide in methanol (to destroy endogenous peroxidases). Sections were then incubated at high temperatures for 30 minutes for antigen retrieval and incubated overnight in blocking solution with horse anti-human MMP1 antibody (1:200 R&D Systems, Minneapolis, MN) at 4 degrees C. Sections were washed with TBST (150mmol/L NaCl, 10mmol/L Tris, and 0.05% Tween 20) and incubated with biotinylated anti-horse IgG (Vector Laboratories, Burlingame, CA) for 1 hour. Labeled cells were visualized using an avidin-biotin peroxidase complex (Vectastain Avidin-Biotin Complex kit, Vector Laboratories, Burlingame, CA) and 3,3’-diaminobenzidine (DAB, Sigma-Aldrich, St. Louis, MO). Sections were counterstained with hematoxylin. Positive human MMP1 staining within lungs and tumors were assessed. Masson Trichrome Staining was performed using their manufacture’s protocol (Thermo Scientific, Kalamazoo, MI).

Morishita A., Gerber A., Gow C.H., Zelonina T., Chada K, & D’Armiento J. (2018). Cell Specific Matrix Metalloproteinase-1 Regulates Lung Metastasis Synergistically with Smoke Exposure. Journal of cancer research forecast, 1(2), 1014.

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Immunohistochemistry of pCDK4 in Xenografts

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Formalin-fixed, paraffin-embedded tissue sections of HCC1954 and MDA-MB-468 xenografts were deparaffinized, boiled in 10 mM sodium citrate (pH 6.0) for 10 min, and treated with 3% H₂O₂ for 10 min. Following, slides were blocked with 10% goat serum in tris-buffered saline with 0.1% Tween 20 and 1% BSA and incubated with pCDK4 Thr¹⁷² antibody overnight at 4°C in tris-buffered saline with 0.1% Tween 20, 5% goat serum, and 1% BSA. pCDK4 Thr¹⁷² antibody (STJ29359, St John’s Laboratory) was used at a dilution of 1:1000. Subsequently, slides were incubated with a biotinylated secondary antibody diluted in tris-buffered saline with 0.1% Tween 20 and 1% BSA for 30 min at RT. Antibody was visualized using the VECTASTAIN Avidin-Biotin Complex Kit (PK-4000, Vector Laboratories) and ImmPACT diaminobenzidine substrate (SK-4105, Vector Laboratories) and counterstained with Vector Hematoxylin QS (H-3404, Vector Laboratories). All reagents were used according to the manufacturer’s instructions. Subsequently, sections were dehydrated and mounted with Permount.

Fassl A., Brain C., Abu-Remaileh M., Stukan I., Butter D., Stepien P., Feit A.S., Bergholz J., Michowski W., Otto T., Sheng Q., Loo A., Michael W., Tiedt R., DeAngelis C., Schiff R., Jiang B., Jovanovic B., Nowak K., Ericsson M., Cameron M., Gray N., Dillon D., Zhao J.J., Sabatini D.M., Jeselsohn R., Brown M., Polyak K, & Sicinski P. (2020). Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition. Science Advances, 6(25), eabb2210.

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Quantification of DNA Synthesis in Cells

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Incorporation of ³H-deoxythymidine (³H-dT, 1uCi/ml) was used to assess DNA synthesis, as described previously [38 (link),42 (link),43 (link)]. Cells were incubated with ³H-dT during the final 4 h of culture. DNA that had incorporated ³H-dT was collected onto glass fiber filters by a semiautomatic cell harvester (Skatron), and the radioligand was assessed by scintillation spectroscopy. To visualize cells synthesizing DNA, GNPs were exposed to the S-phase marker bromodeoxyuridine (BrdU) (10 uM, Sigma) during the final 4 h of incubation. After fixation, cells were either exposed to 2 N HCl (30 min) or pretreated with PBS/0.3% Triton X-100 and then DNaseI (500 U/ml, Worthington) [44 (link)] and subsequently processed for BrdU immunocytochemistry using monoclonal anti-BrdU (1:200; Dako, Carpinteria, CA) and visualized using a Vectastain avidin-biotin complex kit and Vector SG peroxidase substrate with a 3,3′-diaminobenzidine (DAB) chromogen (Vector Laboratories, Burlingame, CA) as previously described [45 (link)]. The labeling index, defined as the proportion of total cells incorporating BrdU into the nucleus, was determined by scoring the cells in five randomly selected, non-overlapping fields in each of the two to four dishes per group per experiment.

Rossman I.T., Lin L., Morgan K.M., DiGiovine M., Van Buskirk E.K., Kamdar S., Millonig J.H, & DiCicco-Bloom E. (2014). Engrailed2 modulates cerebellar granule neuron precursor proliferation, differentiation and insulin-like growth factor 1 signaling during postnatal development. Molecular Autism, 5, 9.

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