Aminoethylcarbazole

Manufactured by Merck Group

Sourced in Germany

Aminoethylcarbazole is a chemical compound used as a substrate in various laboratory applications. It is a colorless to pale yellow crystalline solid. Aminoethylcarbazole is commonly used as a chromogenic substrate in enzyme-linked immunosorbent assays (ELISA) and other immunohistochemical techniques for the detection and visualization of target analytes.

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Lab products found in correlation

Horseradish peroxidase hrp conjugated rabbit anti mouse ig antibody, by Agilent Technologies (4 mentions) Streptavidin horseradish peroxidase conjugate, by Southern Biotech (1 mentions) Immobilon p membrane, by Merck Group (1 mentions) Protein assay reagent, by Bio-Rad (1 mentions) 3 3 diaminobenzidine tetrahydrochloride, by Merck Group (1 mentions) Nanozoomer 2.0 ht scanner, by Hamamatsu Photonics (1 mentions) Vectastain abc kit, by Vector Laboratories (1 mentions) Hrp conjugated rabbit anti mouse ig antibody, by Agilent Technologies (1 mentions) Super block, by ScyTek Laboratories (1 mentions) Goat anti mouse igg1, by Southern Biotech (1 mentions) Oct compound, by Sakura Finetek (1 mentions)

Close spelling variants of this product

Aminoethylcarbazole color reaction (2 citations) Aminoethylcarbazole substrate (2 citations)

9 protocols using aminoethylcarbazole

ELISPOT Assay for IFNγ and IL-5 Secreting Cells

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96-well nitrocellulose-bottomed microplates (Millipore, Billerica, MA) were coated with a rat antibody specific for mouse IFNγ or for mouse IL-5 (BD Pharmigen, Le Pont de Claix, France). After plate washing, freshly isolated splenocytes (2 x 10⁵ cells/well) from immunized and control BALB/c ByJ mice were added to the plate and incubated overnight with murine IL-2 (10 μg/mL; Roche-Boehringer Mannheim, Meylan, France) and either heat-inactivated HSV-2 strain G (10⁵ CCID₅₀/mL equivalents), recombinant HSV gD2 (1 μg/mL; Mybiosource, San Diego, CA), concanavalin A (2.5 μg/mL) as a positive control, or medium as a negative control. The plates were washed and the locations where cells secreting INFγ or IL-5 during incubation were stained with a biotinylated anti-mouse IFNγ or IL-5 antibody (BD Pharmigen, Le Pont de Claix, France), a streptavidin-horseradish peroxidase conjugate (Southern Biotechnology), and a chromogenic substrate (amino ethyl carbazole; Sigma-Aldrich, St. Louis, MO). Plates were read with an automatic ELISPOT reader (Microvision Instruments, France) and stained cells were counted with Spot software (Microvision Instruments, France). Results were expressed as the number of cells (spots) per 10⁶ splenocytes. The threshold of positivity was established at 20 cells/10⁶ splenocytes.

Bernard M.C., Barban V., Pradezynski F., de Montfort A., Ryall R., Caillet C, & Londono-Hayes P. (2015). Immunogenicity, Protective Efficacy, and Non-Replicative Status of the HSV-2 Vaccine Candidate HSV529 in Mice and Guinea Pigs. PLoS ONE, 10(4), e0121518.

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Plasma Viremia Quantification Assay

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Ten days post challenge (p.c.), plasma samples from CB6F1 mice were obtained, and viremia was determined in a focal infectivity assay [56 (link)]. Serial dilutions of plasma were incubated with M. dunni cells for 3 days under standard tissue culture conditions. When cells reached ~100% confluence, they were fixed with ethanol, labeled with F-MuLV Env-specific MAb 720 [48 (link)], and then with a horseradish peroxidase (HRP)-conjugated rabbit antimouse Ig antibody (Dako, Hamburg, Germany). The assay was developed using aminoethylcarbazole (Sigma-Aldrich, Deisenhofen, Germany) as substrate to detect foci. Foci were counted, and focus-forming units (FFU)/ml plasma were calculated.

Kaulfuß M., Wensing I., Windmann S., Hrycak C.P, & Bayer W. (2017). Induction of complex immune responses and strong protection against retrovirus challenge by adenovirus-based immunization depends on the order of vaccine delivery. Retrovirology, 14, 8.

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Quantifying Melanocyte Protein Expression

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Cell lysates were obtained from healthy melanocytes (HM162P7) and also immortalized cell lines PIG1P51 and PIG3VP24; P stands for passage) and protein content was measured using Bio-Rad Protein Assay reagent (Bio-Rad, Hercules, CA, USA), for equal loading at 20 μl for cellular proteins, or 5 μg of purified HSP70 per slot onto a 10% polyacrylamide minigel. After electrophoresis, separated proteins were transferred on to Immobilon-P membrane (Millipore, Billerica, MA, USA). The blots were incubated with SPA-820 to constitutive and inducible HSP70 (mouse monoclonal IgG1; Enzo Life Sciences). In a separate experiment blots were incubated with SPA-811 to inducible HSP70 (polyclonal rabbit; Enzo Life Sciences), and HMB45 to gp100 (monoclonal mouse IgG1; Dako,Carpinteria, CA, USA). After washing, blots were reacted with horseradish peroxidase labeled goat anti-mouse antiserum; Dako) or alkaline phosphatase labeled goat anti-rabbit antiserum Dako) conjugated secondary Abs. Blots were developed with aminoethylcarbazole (Sigma) as the substrate.

Mosenson J.A., Flood K., Klarquist J., Eby J.M., Koshoffer A., Boissy R.E., Overbeck A., C.Tung R, & Poole I.C. (2014). PREFERENTIAL SECRETION OF INDUCIBLE HSP70 BY VITILIGO MELANOCYTES UNDER STRESS. Pigment cell & melanoma research, 27(2), 209-220.

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Quantifying Murine Retrovirus Viremia

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Ten days post challenge (p.c.), plasma samples from CB6F1 mice were obtained, and viremia was determined in a focal infectivity assay [56 (link)]. Serial dilutions of plasma were incubated with M. dunni cells for 3 days under standard tissue culture conditions. When cells reached ~100% confluence, they were fixed with ethanol, labeled with F-MuLV Env-specific MAb 720 [49 (link)], and then with a horseradish peroxidase (HRP)-conjugated rabbit anti-mouse Ig antibody (Dako, Hamburg, Germany). The assay was developed using aminoethylcarbazole (Sigma-Aldrich, Deisenhofen, Germany) as substrate to detect foci. Foci were counted, and focus-forming units (FFU)/ml plasma were calculated.

Bongard N., Le-Trilling V.T., Malyshkina A., Rückborn M., Wohlgemuth K., Wensing I., Windmann S., Dittmer U., Trilling M, & Bayer W. (2019). Immunization with a murine cytomegalovirus based vector encoding retrovirus envelope confers strong protection from Friend retrovirus challenge infection. PLoS Pathogens, 15(9), e1008043.

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Immunohistochemical Staining of Tissue Sections

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Tissues were snap-frozen, cut into 6 µm sections and fixed in acetone/0.02% H₂O₂. For detection of CD3 (Dako Heverlee) and Ki67 (Novocastra), endogenous peroxidases were quenched with 3% H₂O₂ in methanol for 20 min. After microwaving in citrate buffer (10 mM, pH 6.0) for antigen retrieval, sections were blocked for 1 h in 10 mM Tris, 5 mM EDTA, 0.15 M NaCl, 0.05% Tween-20 and 10% normal mouse serum (NMS), before being stained overnight with primary antibodies against CD4 (GK1.5) and CD8 (Lyt2) (both from Dako) at 4 °C in PBS/0.1% BSA. After washing, biotinylated rabbit anti-rat secondary antibody (Dako) or goat anti-rabbit serum was added in PBS/0.1% BSA/2% NMS for 1 h at room temperature. Enzyme activity was revealed by using the Vectastain ABC kit (Vector Laboratories). Aminoethylcarbazole (Sigma) or 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) was used as a chromogen for horseradish peroxidase activity. The sections were counterstained with haematoxylin and mounted with glycerol-gelatin. The slides were scanned with the NanoZoomer 2.0HT scanner (Hamamatsu) and analysed with the NanoZoomer Digital Pathology programme.

Bain C.C., Montgomery J., Scott C.L., Kel J.M., Girard-Madoux M.J., Martens L., Zangerle-Murray T.F., Ober-Blöbaum J., Lindenbergh-Kortleve D., Samsom J.N., Henri S., Lawrence T., Saeys Y., Malissen B., Dalod M., Clausen B.E, & Mowat A.M. (2017). TGFβR signalling controls CD103+CD11b+ dendritic cell development in the intestine. Nature Communications, 8, 620.

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Quantification of Retroviral Infectious Centers

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Twenty-one days post-challenge (p.c.), animals were sacrificed by cervical dislocation, the spleens were removed and weighed, and single-cell suspensions were prepared. Serial dilutions of isolated spleen cells were seeded onto M. dunni cells, and cells were incubated under standard tissue culture conditions for 3 days. When cells reached ~100% confluence, they were fixed with ethanol and labeled with F-MuLV Env-specific monoclonal antibody MAb 720 (32 (link)) and then with a horseradish peroxidase (HRP)-conjugated rabbit anti-mouse Ig antibody (Dako, Hamburg, Germany). The assay was developed using aminoethylcarbazole (Sigma-Aldrich, Deisenhofen, Germany) as substrate to detect foci. The resulting foci were counted, and infectious centers (IC)/10⁸ spleen cells were calculated.

Podschwadt P., Malyshkina A., Windmann S., Papadamakis A., Kerkmann L., Lapuente D., Tenbusch M., Lu M., Schindler M., Lang K.S., Hansen W, & Bayer W. (2022). Immune suppression of vaccine-induced CD8+ T-cell responses by gamma retrovirus envelope is mediated by interleukin-10-producing CD4+ T cells. Frontiers in Immunology, 13, 934399.

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Viral Load Quantification by IC Assay

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Determination of viral loads by IC assay was performed as described previously (36 (link)). Spleens were cropped and rinsed with RPMI containing 10% FCS and 50 µg mL⁻¹ penicillin/streptomycin. Spleen cells were counted and serially diluted before seeding them onto Mus dunni tail fibroblast cells and incubated under standard tissue culture conditions for 3 days, fixed with ethanol and labeled with the primary F-MuLV Env-specific MAb 720 (37 (link)). After washing, a secondary horseradish peroxidase (HRP)-conjugated rabbit anti-mouse Ig antibody (Dako) was added. Foci representing ICs were detectable after adding of aminoethylcarbazole (Sigma-Aldrich) as substrate for HRP. Foci were counted and ICs/spleen values were calculated.

Knuschke T., Rotan O., Bayer W., Kollenda S., Dickow J., Sutter K., Hansen W., Dittmer U., Lang K.S., Epple M., Buer J, & Westendorf A.M. (2018). Induction of Type I Interferons by Therapeutic Nanoparticle-Based Vaccination Is Indispensable to Reinforce Cytotoxic CD8+ T Cell Responses During Chronic Retroviral Infection. Frontiers in Immunology, 9, 614.

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Quantifying Retroviral Infectious Centers

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Determination of viral loads by infectious-center assay was performed as described previously (55 (link)). Spleens were cropped and rinsed with RPMI medium containing 10% FCS and 50 μg ml⁻¹ penicillin/streptomycin. Spleen cells were counted and serially diluted before they were seeded onto Mus dunni cells, and then they were incubated under standard tissue culture conditions for 3 days, fixed with ethanol, and labeled with the primary F-MuLV Env-specific monoclonal antibody 720 (56 (link)). After the cells were washed, a secondary horseradish peroxidase (HRP)-conjugated rabbit anti-mouse Ig antibody (Dako) was added. Foci representing infectious centers were detectable after the addition of aminoethylcarbazole (Sigma-Aldrich) as the substrate for HRP. Foci were counted and numbers of infectious centers (IC) per spleen were calculated.

Knuschke T., Kollenda S., Wenzek C., Zelinskyy G., Steinbach P., Dittmer U., Buer J., Epple M, & Westendorf A.M. (2021). A Combination of Anti-PD-L1 Treatment and Therapeutic Vaccination Facilitates Improved Retroviral Clearance via Reactivation of Highly Exhausted T Cells. mBio, 12(1), e02121-20.

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Immunohistochemical Analysis of Vitiligo Skin

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Four-millimeter punch biopsies were obtained from non-vitiligo control patients, or non-lesional and lesional areas of actively progressing vitiligo in consenting patients attending the Loyola Dermatology outpatient clinic. The skin samples were snap-frozen in OCT compound (Sakura Finetek, Torrence, CA, USA). Cryostat sections (8 mm) were fixed in cold acetone, and indirect immunoperoxidase staining procedures were performed essentially as described previously (Le Poole et al., 1993b (link)). Briefly, tissue sections were treated with Super Block (ScyTek, West Logan, UT, USA) to prevent nonspecific antibody binding, and incubated with primary antibodies SPA-810 to human HSP70i (mouse monoclonal; Enzo Life Sciences formerly Stressgen, Farmingdale, NY, USA), followed by horseradish peroxidase–conjugated secondary antibodies (goat anti-mouse IgG1; Southern Biotech, Birmingham, AL, USA). Enzymatic detection was finalized with aminoethylcarbazole as a substrate (Sigma). Images were imported using Adobe Photoshop software (Adobe Systems Inc., San Jose, CA, USA). Immunohistochemical analysis was performed at least in triplicate for each staining and on each tissue sample, on at least three sections. Actual sample sizes are listed in the figure legends.

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