Vector blue substrate

Manufactured by Vector Laboratories

Sourced in United States

Vector Blue is a chromogenic substrate that is used for the detection and visualization of enzymatic activity in various biological applications. It is a sensitive and specific substrate that produces a blue-colored reaction product when cleaved by the target enzyme. Vector Blue can be used in a wide range of assays, including immunohistochemistry, in situ hybridization, and enzyme-linked immunosorbent assays (ELISAs).

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Lab products found in correlation

Streptavidin alkaline phosphatase, by Vector Laboratories (3 mentions) Multiscreen filter plate, by Merck Group (2 mentions) Poly l lysine (pll), by Merck Group (2 mentions) Salmon sperm dsdna, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Merck Group (2 mentions) L glutamine, by Merck Group (2 mentions) Multiscreen hts 96 well elispot plates, by Merck Group (2 mentions) 70 μm falcon cell strainer, by BD (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Biotin conjugated goat anti mouse igg, by Agilent Technologies (1 mentions) Streptavidin ap, by Vector Laboratories (1 mentions) Rpmi 1640, by Harvard Bioscience (1 mentions) Axio scan z1 slide scanner, by Zeiss (1 mentions) Avidin biotin blocking kit, by Vector Laboratories (1 mentions) Permafluor mounting medium, by Thermo Fisher Scientific (1 mentions) Clone fjk 16s, by Abcam (1 mentions) Citrate buffer, by Vector Laboratories (1 mentions) Normal goat serum (ngs), by Biosera (1 mentions) Dab working solution, by Vector Laboratories (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Vector Blue (44 citations) Vector Blue Alkaline Phosphatase Substrate Kit (32 citations) Vector Blue Alkaline Phosphatase Substrate Kit III (29 citations) Vector Blue Substrate Kit (25 citations) Vector Blue AP Substrate Kit III (5 citations) Vector blue alkaline phosphatase (Blue AP) substrate kit (4 citations) Vector Blue Alkaline Phosphatase Substrate (3 citations) Vector Blue AP substrate (2 citations) Vector Blue substrate kit III (2 citations) ABC-AP Vector Blue Substrate reagents (2 citations)

8 protocols using vector blue substrate

Quantifying Cell-Derived Immune Complexes

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Multiscreen Filter plates (Millipore) were coated with Poly-L-lysine (Sigma Aldrich), and subsequently with salmon sperm dsDNA (Invitrogen) or SmRNP (Arotec Diagnostics). Nucleosome plates were generated via incubation with dsDNA followed by histone (Sigma Aldrich). Plates were blocked in 5% FCS in PBS and then plated with 1 million splenocytes or bone marrow cells for serial dilution. Detection was performed by incubation with anti-IgG-biotin (Jackson Immunoresearch) followed by streptavidin-alkaline phosphatase (Vector Laboratories). Development was performed with Vector Blue substrate (Vector Labs).

Schell S.L., Bricker K.N., Fike A.J., Chodisetti S.B., Domeier P.P., Choi N.M., Fasnacht M.J., Luckenbill S.A., Ziegler S.F, & Rahman Z.S. (2021). Context-dependent miR-21 regulation of TLR7-mediated autoimmune and foreign antigen driven antibody-forming cell and germinal center responses. Journal of immunology (Baltimore, Md. : 1950), 206(12), 2803-2818.

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ELISPOT Analysis of Antigen-Specific Immune Cells

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Inguinal draining lymph nodes of mCC1- (n = 8) or mIgG1- (n = 7) treated MP4-immunized mice were collected on day 27.6 ± 5.4 after disease onset, disintegrated mechanically and filtered through a 70 μm Falcon cell strainer (BD Biosciences). Cells were washed twice with RPMI-1640 (Biochrom AG) and resuspended in HL-1 (Lonza) containing 1% L-glutamine (Sigma) and 1% penicillin/streptomycin (Sigma). MultiScreen^®HTS 96-well ELISPOT plates (Merck Millipore) were coated in duplicate wells with either 10 μg/ml MP4 or 15 μg/ml anti-mouse IgG (MabTech). PBS alone (Biochrom) served as negative control coating. Plates were blocked with 10% fetal bovine serum (FBS; Gibco) in sterile PBS at room temperature for 2 h. Cells were plated at 1 × 10⁶ cells/well and incubated at 37 °C and 7% CO₂ for 24 h. Biotin-conjugated goat anti-mouse IgG (Dako) was used as secondary antibody at 1:2,000 dilution in 0.5% FBS/PBS. After incubation with Streptavidin-AP (Vector Laboratories) at 1:800 dilution in 0.5% FBS/PBS for 2 h, plates were developed with Vector Blue substrate (Vector Laboratories). The spots were counted on an ImmunoSpot Series 6 UV Analyzer (CTL-Europe).

Rovituso D.M., Scheffler L., Wunsch M., Kleinschnitz C., Dörck S., Ulzheimer J., Bayas A., Steinman L., Ergün S, & Kuerten S. (2016). CEACAM1 mediates B cell aggregation in central nervous system autoimmunity. Scientific Reports, 6, 29847.

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Multiplex Immunohistochemistry for Intestinal Immune Cells

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Paraffin-embedded longitudinal sections of the gut were cleared and rehydrated in xylene and graded alcohols, followed by antigen retrieval through pressure boiling in citrate buffer (Vector, Burlingame, CA, USA). Endogenous peroxidase activity was blocked with 3% H₂O₂ (Fisher Scientific) in dH₂O, 10% normal goat serum (Biosera, Boussens, France) in PBS was used to block unspecific binding sites, and endogenous avidin/biotin was blocked using an avidin-biotin blocking kit according to manufacturer’s instructions (Vector). Samples were stained with biotinylated rat anti-mouse Foxp3 antibody (eBioscience, clone FJK-16s) and purified rabbit anti-mouse CD3 antibody (Abcam, polyclonal). Samples were then incubated with streptavidin-coupled alkaline phosphatase (Vector) for 1 h at room temperature, washed, and Vector Blue substrate (SK-5300, Vector) added according to manufacturer’s instructions. Samples were subsequently incubated with horseradish peroxidase-coupled goat anti-rabbit antibody, washed, and DAB working solution added according to manufacturer’s instructions (Vector). Slides were mounted in Permafluor mounting medium (Thermo Scientific) prior to digitalization with an Axioscan.Z1 slide scanner (Carl Zeiss).

Mair I., Zandee S.E., Toor I.S., Saul L., McPherson R.C., Leech M.D., Smyth D.J., O’Connor R.A., Henderson N.C, & Anderton S.M. (2018). A Context-Dependent Role for αv Integrins in Regulatory T Cell Accumulation at Sites of Inflammation. Frontiers in Immunology, 9, 264.

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ELISPOT Assay for Murine Lymphocytes

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MultiScreen®HTS 96-well ELISPOT plates (Merck Millipore, Darmstadt, Germany) were coated overnight with either 10 μg/ml MP4 (Alexion Pharmaceuticals, Inc.) or 15 μg/ml anti-mouse IgG (MabTech, Nacka Strand, Sweden), while coating with sterile PBS (Sigma-Aldrich) served as a negative control. Plates were blocked with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in sterile PBS at room temperature for 2 h. Inguinal lymph nodes were disintegrated mechanically and filtered through a 70 μm cell strainer (Corning Inc., Corning, NY, USA). Cells were washed twice with complete RPMI-1640 medium (Gibco), subsequently resuspended in HL-1 medium (Lonza, Basel, Switzerland) containing 1% L-glutamine (Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich) and plated at 1 × 10⁶ cells/well followed by incubation at 37 °C and 7% CO₂ for 24 h. Biotinylated goat anti-mouse IgG (Dako, Glostrop, Denmark) served as secondary antibody at 1:2000 dilution in 0.5% FBS/PBS + 0.025% Tween at 4 °C overnight. Plates were washed and incubated with streptavidin-alkaline phosphatase (Vector) at 1:800 dilution in 0.5% FBS/PBS for 2 h. Vector Blue substrate (Vector) was used for development. The spots were counted on an ImmunoSpot Series 6 UV Analyzer (CTL-Europe, Bonn, Germany).

Bail K., Notz Q., Rovituso D.M., Schampel A., Wunsch M., Koeniger T., Schropp V., Bharti R., Scholz C.J., Foerstner K.U., Kleinschnitz C, & Kuerten S. (2017). Differential effects of FTY720 on the B cell compartment in a mouse model of multiple sclerosis. Journal of Neuroinflammation, 14, 148.

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Histological Analysis of gWAT Adipocytes

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Tissues were fixed in 4% formalin and embedded in paraffin. Deparaffinized tissue sections (4 µmol/L) were stained with hematoxylin-eosin. Immunohistochemistry was performed with rabbit anti-BNIP3, followed by alkaline phosphatase–conjugated goat antirabbit IgG. Bound alkaline phosphatase activity was detected using Vector Blue substrate (Vector Laboratories, Burlingame, CA) in the presence of levamisole (1 mmol/L). gWAT cell size was determined by acquiring images from nonoverlapping fields, followed by Adiposoft analysis. The average area and diameter of >200 cells were determined and expressed in arbitrary units.

Tol M.J., Ottenhoff R., van Eijk M., Zelcer N., Aten J., Houten S.M., Geerts D., van Roomen C., Bierlaagh M.C., Scheij S., Hoeksema M.A., Aerts J.M., Bogan J.S., Dorn GW 2.n.d., Argmann C.A, & Verhoeven A.J. (2016). A PPARγ-Bnip3 Axis Couples Adipose Mitochondrial Fusion-Fission Balance to Systemic Insulin Sensitivity. Diabetes, 65(9), 2591-2605.

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Multiscreen Assay for Antigen-Specific IgG/IgA

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Multiscreen_HTS HA plates (Millipore, MSHAN4510) were coated with 5μg/mL F protein antigen (Sino Biological Inc), 10μg/mL Human Serum Albumin (HSA, Sigma), 5μg/mL tetanus toxoid protein (Statens Seruminstitute) and 10μg/mL polyvalent goat anti-human immunoglobulins (Caltag). After washing, plates were blocked with 1% skimmed milk for 45 minutes at 37°C before 100μL/well PBMCs in R10 (RPMI, 10% Foetal Bovine Serum, 2mM L-Glutamine, 50μg/ml Streptomycin, 50U Penicillin) were added at a starting dilution of 2×10⁶/mL and incubated overnight at 37°C, 5% CO₂, 95% humidity. After washing plates were developed anti-human IgG-FITC (Sigma) and anti-human IgA-Biotin (AbD Serotec). After washing anti-FITC AP (Sigma) and Streptavidin-HRP (AbD Serotec) were added for 30 minutes at room temperature. Following final washes 3-Amino-9-ethylcarbazole (AEC) substrate kit (Sigma) was added for 30 minutes at room temperature, washed with dH₂O and 100μL/well Vector Blue substrate (Vector Laboratories) added for 10 minutes at room temperature before a final wash in dH₂O. After drying overnight plates were read using Autoimmun Diagnostika (AID version 5.0) and responses measured as the antigen-specific spots per million PBMCs with HSA background subtracted. A positive response was defined as any detection of spots above HSA background.

Green C., Scarselli E., Sande C., Thompson A., de Lara C., Taylor K., Haworth K., Del Sorbo M., Angus B., Siani L., Di Marco S., Traboni C., Folgori A., Colloca S., Capone S., Vitelli A., Cortese R., Klenerman P., Nicosia A, & Pollard A. (2015). Chimpanzee adenovirus and MVA-vectored respiratory syncytial virus vaccine is safe and expands humoral and cellular immunity in adults. Science translational medicine, 7(300), 300ra126.

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Immunohistochemical Analysis of Rat Thymus

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Sections of freshly frozen tissues and cytosmears were fixed in acetone and immunostained as described previously [23] (link)–[24] (link). For light microscopy, molecules of interest were stained blue using alkaline phosphatase-conjugated secondary/tertiary antibodies and Vector Blue substrate (Vector Laboratories, Burlingame, CA, USA). Additionally, type IV collagen, which reveals the framework of tissues was stained brown with peroxidase-conjugated secondary antibody and 3, 3′-diaminobenzidine substrate (Dojindo Molecular Technologies, Kumamoto, Japan). Photomicrographs were captured with a Microphot-FX microscope (Nikon, Tokyo, Japan) and a DP26 digital camera (Olympus, Tokyo, Japan). The proportions of the cortical area and two medullary subareas (described in Results) were calculated in the rat thymus with digital image analysis software (cellSens, Olympus). Thymi from three male Lewis rats were used for this purpose.
For fluorescence microscopy, fluorescent dye-conjugated secondary antibodies or streptavidin were used. Multichannel color fluorescence images were captured with an Axioskop 2 Plus fluorescence microscope equipped with an AxioCam MRm or MRc5 camera (Zeiss, Oberkochen, Germany). We assigned pseudocolors to each channel to make merged images more comprehensible by maximizing contrast using AxioVision software (Zeiss).

Sawanobori Y., Ueta H., Dijkstra C.D., Park C.G., Satou M., Kitazawa Y, & Matsuno K. (2014). Three Distinct Subsets of Thymic Epithelial Cells in Rats and Mice Defined by Novel Antibodies. PLoS ONE, 9(10), e109995.

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Quantifying Lymphocyte Binding Assay

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Multiscreen Filter plates (Millipore) were coated with Poly-L-lysine (Sigma Aldrich), and subsequently with salmon sperm dsDNA (Invitrogen) or SmRNP (Arotec Diagnostics).
Nucleosome plates were generated via incubation with dsDNA followed by histone (Sigma Aldrich). Plates were blocked in 5% FCS in PBS and then plated with 1 million splenocytes or bone marrow cells for serial dilution. Detection was performed by incubation with anti-IgGbiotin (Jackson Immunoresearch) followed by streptavidin-alkaline phosphatase (Vector Laboratories). Development was performed with Vector Blue substrate (Vector Labs).

Schell S.L., Bricker K.N., Fike A.J., Chodisetti S.B., Domeier P.P., Choi N., Fasnacht M.J., Luckenbill S.A., Ziegler S.F., & Rahman Z.S.M. (2021). Context-dependent miR-21 regulation of TLR7-mediated autoimmune and foreign antigen driven antibody-forming cell and germinal center responses.

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