Vector red alkaline phosphatase substrate

Manufactured by Vector Laboratories

Sourced in United States, United Kingdom

Vector Red alkaline phosphatase substrate is a chromogenic substrate used for the detection and visualization of alkaline phosphatase activity in biological samples. It produces a red-colored reaction product upon enzymatic conversion.

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Lab products found in correlation

Ap live stain, by Thermo Fisher Scientific (2 mentions) Alkaline phosphatase conjugated anti digoxigenin antibody, by Merck Group (1 mentions) Mircury lna mirna ish optimization kit, by Qiagen (1 mentions) Vector blue alkaline phosphatase substrate, by Vector Laboratories (1 mentions) Benchmark xt, by Roche (1 mentions) Cd68 kp 1, by Roche (1 mentions) Cd34 qbend 10, by Roche (1 mentions) Cd61 2f 2, by Cell Marque (1 mentions) Hematoxylin and eosin, by Merck Group (1 mentions) Anti inos antibody sc 651, by Santa Cruz Biotechnology (1 mentions) Anti galectin 3 af1197, by R&D Systems (1 mentions) Dig labeling mix, by Roche (1 mentions) Anti dig antibody conjugated to alkaline phosphatase, by Roche (1 mentions) Dp70 microscope, by Olympus (1 mentions) Sars cov 2 nucleocapsid rabbit polyclonal antibody, by Thermo Fisher Scientific (1 mentions) Vectastain abc ap, by Vector Laboratories (1 mentions) Haematoxylin qs, by Vector Laboratories (1 mentions) Anti mouse cd68, by Bio-Rad (1 mentions) Anti mouse ly6g, by BD (1 mentions) Levamisole solution, by Vector Laboratories (1 mentions)

Spelling variants of this product

Vector Red Alkaline Phosphatase Substrate Kit (39 citations) Vector Red (36 citations) Vector Red substrate (18 citations) Vector Red Alkaline Phosphatase Substrate Kit I (15 citations) ImmPACT Vector Red Alkaline Phosphatase Substrate (5 citations) Alkaline Phosphatase (4 citations) Alkaline phosphatase red substrate (3 citations) Alkaline phosphatase substrate (3 citations) VECTOR® Red Alkaline Phosphatase (Red AP) Substrate Kit (3 citations) Vector Red alkaline phosphatase substrate solution (2 citations)

17 protocols using vector red alkaline phosphatase substrate

miR-182 LNA In Situ Hybridization

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The protocol from the miRCURY LNA miRNA ISH optimization kit (Exiqon, Vedbaek, Denmark) was followed with modifications. Formalin-fixed, paraffin-embedded TMA sections (5 μm) were placed onto hydrophilic slides, baked overnight at 60°C, deparaffinized, and incubated for 20 minutes at 37°C with 15 μg/mL of proteinase K for protein digestion. Digoxigenin-labeled miR-182 LNA probe (80 nmol/L), digoxigenin-labeled U6 LNA probe (10 nmol/L) (positive control), or no probe (negative control) was incubated at 48°C for 60 minutes followed by stepwise 5-minute washes in saline-sodium citrate buffer at 42°C (×1, ×−0.5, ×−0.2, and then ×0.2) at room temperature. Slides were blocked and incubated for 60 minutes with alkaline phosphatase–conjugated anti-digoxigenin antibody (Sigma-Aldrich, St. Louis, MO) at 1:200. Alkaline phosphatase was visualized with Vector Red alkaline phosphatase substrate (Vector Laboratories, Burlingame, CA) for 90 minutes and stopped with KTBT buffer. Slides were counterstained with DAPI.

Baumann B., Acosta A.M., Richards Z., Deaton R., Sapatynska A., Murphy A., Kajdacsy-Balla A., Gann P.H, & Nonn L. (2019). Association of High miR-182 Levels with Low-Risk Prostate Cancer. The American Journal of Pathology, 189(4), 911-923.

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Double Immunostaining of GFAs and Aβ

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To examine whether GFAs occur in association with Aβ deposits, double immunostaining with AT8 and 12B2 was done in all cases that had Aβ deposits in the frontal cortex, striatum and/or amygdala. Primary antibody labeling in the first cycle (AT8) was detected in the same way as single staining using the DAB reaction. Then, primary antibody labeling in the second cycle (12B2) was also detected in the same way as single staining, and the reaction was visualized with Vector Blue Alkaline Phosphatase Substrate (Vector Laboratories, Inc.) as the chromogen.
Double staining using the Gallyas method and AT8 immunohistochemistry was also done in representative AGD and PSP cases to examine whether GFAs actually have TA‐like Gallyas‐positive glial threads. Sections were first stained by the Gallyas method, followed by immunostaining with AT8. The peroxidase labeling was visualized with Vector Red Alkaline Phosphatase Substrate (Vector Laboratories, Inc.) as the chromogen. Sections were lightly counterstained with hematoxylin.

Miki T., Yokota O., Haraguchi T., Ishizu H., Hasegawa M., Ishihara T., Ueno S., Takenoshita S., Terada S, & Yamada N. (2020). Factors associated with development and distribution of granular/fuzzy astrocytes in neurodegenerative diseases. Brain Pathology, 30(4), 811-830.

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Immunocytochemistry and Immunohistochemistry of Meningiomas

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For immunocytochemistry, cytospin preparations of BMEN1 cells were analyzed by the OHSU Department of Pathology using Ventana V9 anti-Vimentin on a Vantan XT instrument according to the manufacturer standard protocol (Ventana Medical Systems, Tucson, AZ). For immunohistochemistry of meningiomas and medulloblastomas and associated meninges, 7-micron sections were prepared from formalin-fixed, paraffin-embedded tissues. Deparaffinized sections were blocked and treated with antibodies to MMP9 (EP1255Y from Novus Biologicals, Littleton, CO), Vimentin (V9 from Ventana), CD68 (KP-1 from Ventana), Collagen IV (CIV-22 from Cell Marque, Rocklin, CA), CD34 (QBEND-10 from Ventana), factor VIII (rabbit polyclonal antibody from Cell Marque), or CD61 (2F-2 from Cell Marque). MMP9 was used at 1:500 dilution and developed by hand using Vector Red alkaline phosphatase substrate (Vector Laboratories, Burlingame, CA). All other immunohistochemical stains were performed on a Ventana Benchmark XT autostainer according to protocols specifiied by the manufacturer, and developed using diaminobenzidine chromogen.

Svalina M.N., Kikuchi K., Abraham J., Lal S., Davare M.A., Settelmeyer T.P., Young M.C., Peckham J.L., Cho Y.J., Michalek J.E., Hernandez B.S., Berlow N.E., Jackson M., Guillaume D.J., Selden N.R., Bigner D.D., Nazemi K.J., Green S.C., Corless C.L., Gultekin S., Mansoor A., Rubin B.P., Woltjer R, & Keller C. (2016). IGF1R as a Key Target in High Risk, Metastatic Medulloblastoma. Scientific Reports, 6, 27012.

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SDF-1 and CXCR4 Expression in IPF Lungs

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Human IPF lung specimens were recovered after surgical biopsies performed for diagnostic purpose (n=6). Three mm thick paraffin embedded sections were stained with hematoxylin and eosin to evaluate parenchyma and vessel morphology. Subsequently, the expression of SDF-1 and CXCR4 was evaluated using immunohistochemistry. For this purpose, sections were stained with anti-human SDF-1 (dilution 1:60; monoclonal mouse IgG1, clone 79018; R&D Systems, Lille, France) or anti-human CXCR4 antibody (dilution 1:100; monoclonal mouse IgG2B Clone 44716; R&D Systems). Biotinylated anti-mouse IgG (Abcam, Paris, France, reference ab64255) was used for primary antibody detection and Vector Red alkaline phosphatase substrate (Vector, Philadelphia, reference Sk-5100) was the chromogen. Tissues obtained after lung resection for a localized tumor served as controls.

Smadja D.M., Dorfmüller P., Bieche I., Guerin C., Badoual C., Boscolo E., Kambouchner M., Cazes A., Mercier O., Humbert M., Gaussem P., Bischoff J, & Israël-Biet D. (2014). Cooperation between human fibrocytes and endothelial colony forming cells increases angiogenesis via CXCR4 pathway. Thrombosis and haemostasis, 112(5), 1002-1013.

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Histological Analysis of TB-Infected Lungs

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For TB infected lungs, histology mice were sacrificed by cervical dislocation, a slit was created in the trachea and one lung lobe placed in PBS for confirmation of CFU. A tube was inserted in the trachea and lungs were inflated with 1 ml of 10% Neutral Buffered Formalin (NBF; Sigma Aldrich, UK). The lungs were removed and placed in 10 ml of NBF overnight at 4 °C and then transferred into 70% ethanol and stored at 4 °C.
Paraffin-embedded tissue samples were sectioned (7 μm) and stained with hematoxylin and eosin (Sigma) and Acid Fast stain (BD Biosciences). Immunohistochemical staining was performed using anti-iNOS antibody (sc-651, Santa Cruz, 1/500) and anti-Galectin 3 (AF1197, R&D Systems, 1/500). This was followed by a secondary biotinylated antibody (Vector Laboratories, UK) then avidin–biotin–AP complex and visualized with Vector Red alkaline phosphatase substrate (Vector Laboratories, UK).

McNeill E., Stylianou E., Crabtree M.J., Harrington-Kandt R., Kolb A.L., Diotallevi M., Hale A.B., Bettencourt P., Tanner R., O’Shea M.K., Matsumiya M., Lockstone H., Müller J., Fletcher H.A., Greaves D.R., McShane H, & Channon K.M. (2018). Regulation of mycobacterial infection by macrophage Gch1 and tetrahydrobiopterin. Nature Communications, 9, 5409.

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Double-label In Situ Hybridization Protocol

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Double label ISH was performed as described previously (Di Giorgio et al., 2014 (link); Kim et al., 2013 (link); Poling et al., 2012 (link); Robertson et al., 2009 (link)). Briefly, slide-mounted brain sections were treated similarly to single-label ISH with the following modifications. Digoxigenin (DIG)-labeled antisense mouse Gnrh, Kiss1, Tac2, or Rfrp cRNA were synthesized with DIG labeling mix (Roche). Radiolabeled (³³P) antisense c-fos or Kiss1r (0.05 pmol/ml) and DIG-labeled (1:500) riboprobes were combined with tRNA, heat denatured, and dissolved together in hybridization buffer. The probe mix was applied to slides (100 µl/slide) and hybridized at 55 °C overnight. After the 62 °C washes on day 2, slides were incubated in blocking buffer for 1 h at room temperature and then incubated overnight at room temperature with anti-DIG antibody conjugated to alkaline phosphatase [(Roche) diluted 1:500]. Slides were then washed with Buffer 1 and incubated with Vector Red alkaline phosphatase substrate (Vector Labs, CA) for 1 h at room temperature. The slides were then air-dried, dipped in emulsion, stored at 4 °C, and developed 7–10 days later, depending on the assay.

Semaan S.J, & Kauffman A.S. (2014). Daily successive changes in reproductive gene expression and neuronal activation in the brains of pubertal female mice. Molecular and cellular endocrinology, 0, 84-97.

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SARS-CoV-2 Nucleocapsid Protein Detection

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Tissue sections were stained with SARS-CoV-2 nucleocapsid rabbit polyclonal antibody (Thermo Fisher Scientific). The sections were treated with antigen retrieval solution in a microwave oven and blocked with normal rabbit serum in PBS (pH 7.4). They were then incubated with the rabbit antibody against SARS-CoV-2 NP (1:100 dilution), and subsequently treated with biotin-labelled goat anti-rabbit immunoglobulin (Vector Laboratories, Burlingame, CA, USA), and Vectastain ABC-AP (Vector Laboratories) and Vector Red alkaline phosphatase substrate (Vector Laboratories). The labelled lung sections were counterstained with haematoxylin QS (Vector Laboratories) and observed under an Olympus DP70 microscope (Olympus Corporation).

Seo S.H, & Jang Y. (2020). Cold-Adapted Live Attenuated SARS-Cov-2 Vaccine Completely Protects Human ACE2 Transgenic Mice from SARS-Cov-2 Infection. Vaccines, 8(4), 584.

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Alkaline Phosphatase Staining of mESCs

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mESCs were cultured for 3 d and ALP was detected using Vector Red alkaline phosphatase substrate (Vector Laboratories, Burlingame, CA, USA). Stain solution was prepared according to the manufacturer’s instructions, and then washed with 200-mM Tris-HCl, pH 8.2–8.5 buffer (Wako, JAPAN) and then the stain solution added to the wells. The samples were incubated with the substrate working solution for 30 min in the dark at room temperature. Finally, the cells were washed with Tris-HCl buffer for 5 min and rinsed in water.

Yumoto M., Hemmi N., Sato N., Kawashima Y., Arikawa K., Ide K., Hosokawa M., Seo M, & Takeyama H. (2020). Evaluation of the effects of cell-dispensing using an inkjet-based bioprinter on cell integrity by RNA-seq analysis. Scientific Reports, 10, 7158.

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Alkaline Phosphatase Staining for iPSC Identification

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Alkaline Phosphatase (AP) staining was used to identify the iPSC clones 14 to 21 days after Sendai virus-mediated reprogramming. The AP live stain (Molecular Probes, Eugene, OR) was conducted on clones selected for further propagation and characterization according to the manufacturer’s protocol. The Vector Red Alkaline Phosphatase substrate (Vector Laboratories, Burlingame, CA) was used to stain all other putative iPSC clones to calculate the reprogramming efficiency. In the latter case, cells were fixed with 4% paraformaldehyde for 5 minutes and then incubated with the substrate working solution prepared in 100 mM Tris-HCl at pH 8.5 for 20 minutes at room temperature. The result was checked under microscope after removing the substrate and washing cells with PBS. Colonies with strong red stain in more than 50% of cells were counted as healthy iPSC clones.

Wang K., Guzman A.K., Yan Z., Zhang S., Hu M.Y., Hamaneh M.B., Yu Y.K., Tolu S., Zhang J., Kanavy H.E., Ye K., Bartholdy B, & Bouhassira E.E. (2019). Ultra-High-Frequency Reprogramming of Individual Long-Term Hematopoietic Stem Cells Yields Low Somatic Variant Induced Pluripotent Stem Cells. Cell reports, 26(10), 2580-2592.e7.

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Immunohistochemical Analysis of Aortic Inflammation

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Mouse aortic sinus was serially cut in 7 μm transversal sections, as previously described^{11 (link),68 (link)}. Sections from mouse specimens were fixed in acetone and immunostained with specific antibodies anti-mouse CD68 (macrophages, ABD Serotec, Düsseldorf, Germany), anti-mouse Ly-6G (neutrophils, BD PharmingenTM, San Jose, CA, USA), anti-mouse MMP-9 (R&D Systems). Vector Red alkaline phosphatase substrate: SK-5100; in association with Levamisole solution: SP-5000; which produce a magenta coloration is used for revelations (Vector Laboratories, INC, CA, USA). Quantifications were performed using the MetaMorph or Definiens software. Results for other parameters were calculated as percentages of stained area on total lesion area, number of infiltrating cells per mm2 of lesion area or number of lymphatic vessels in adventitia.

Santiago-Raber M.L., Montecucco F., Vuilleumier N., Miteva K., Baptista D., Carbone F., Pagano S., Roth A., Burger F., Mach F, & Brandt K.J. (2020). Atherosclerotic plaque vulnerability is increased in mouse model of lupus. Scientific Reports, 10, 18324.

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