Aec substrate

Manufactured by Abcam

Sourced in United Kingdom

AEC substrate is a chromogenic substrate used for the detection of target proteins in immunohistochemical (IHC) and immunocytochemical (ICC) applications. It produces a reddish-brown precipitate at the site of the target antigen, allowing for visual identification.

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Lab products found in correlation

Anti rabbit peroxidase conjugated secondary antibody, by Merck Group (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Bx43 upright microscope, by Olympus (2 mentions) Avidin block, by Vector Laboratories (1 mentions) Biotin block, by Vector Laboratories (1 mentions) Q color5 imaging system, by Olympus (1 mentions)

3 protocols using aec substrate

CIGB-325 Inhibits SARS-CoV-2 Infection in MDBK Cells

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MDBK cells were plated on eight-well glass slides and incubated overnight at 37 °C and 5% CO₂. After incubation, cells were pre-treated for 1 h with CIGB-325 (30 μM) or vehicle (PBS) and infected with 100 µL of virus at a concentration of 70,000 TCID₅₀/well (MOI = 0.1). After 1 h of incubation, final volume was completed up to 500 µL, and the appropriate drug’s concentration was maintained for 16 h and 24 h. Subsequently, the cells were washed with cold PBS three times and fixed in 4% formaldehyde for 10 min at 4 °C. After permeabilization with 0.5% Triton X-100 for 10 min, cells were blocked by incubation with 4% bovine serum albumin (Sigma, St Louis, MO, USA) in PBS for 30 min at room temperature, washed again, and incubated with 30 μg/mL human polyclonal anti- SARS-CoV-2 for 2 h at room temperature. Then, peroxidase-conjugated anti-rabbit secondary antibody 1:100 (Sigma, St. Louis, MO, USA) was incubated for 1 h at room temperature and washed 3 times with PBS. Finally, AEC substrate (Abcam, Cambridge, UK) was added and coverglass was mounted using 40% glycerol mounting medium and analyzed using a BX43 upright microscope (Olympus America Inc., Waltham, MA, USA).

Ramón A.C., Pérez G.V., Caballero E., Rosales M., Aguilar D., Vázquez-Blomquist D., Ramos Y., Rodríguez-Ulloa A., Falcón V., Rodríguez-Moltó M.P., Yang K., Perera Y, & Perea S.E. (2022). Targeting of Protein Kinase CK2 Elicits Antiviral Activity on Bovine Coronavirus Infection. Viruses, 14(3), 552.

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Quantifying Macrophages in Aortic Tissue

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5 μM sections of the proximal aorta were fixed in cold acetone and incubated at room temperature in 2% BSA/PBS. Slides were treated with avidin block (Vector, Olean, NY), biotin block (Vector), and peroxidase-activity block (9:1 ratio of methanol:30% H₂O₂). Macrophages were stained using a 1:25 dilution of rat-anti mouse macrophage/monocyte, clone MOMA-2 (MilliporeSigma) in 2% BSA/PBS for 1 hour at RT. Next, slides were stained with a 1:200 dilution of biotin goat-anti rat (BD Biosciences) in 2% BSA/PBS for 30 minutes at 37°C.
Strepavadin-HRP (Biogenex, Fremont, CA) was applied for 20 minutes followed by AEC substrate (Abcam) for 2 minutes. Hematoxylin counterstain was applied for 2 minutes and slides were imaged using a Q-Color5™ imaging system (Olympus, Center Valley, PA). Quantification of macrophage/monocyte area was performed using ImageJ and averaged for four sections.

Marvin J., Rhoads J.P, & Major A.S. (2019). FcγRIIb on CD11c+ cells modulates serum cholesterol and triglyceride levels and differentially affects atherosclerosis in male and female Ldlr−/− mice. Atherosclerosis, 285, 108-119.

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SARS-CoV-2 Infection Inhibition Assay

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MDBK cells were plated on eight well glass slide and incubated overnight at 37 °C and 5% CO2. After incubation, cells were pre-treated for 1 h with CIGB-325 (30 μM) or vehicle (PBS) and infected with 100 µL of virus at a concentration of 70 000 TCID50/well. After 1h of incubation, final volume was completed up to 500 µL and the appropriate drug's concentration was maintained for 16 h and 24 h. Subsequently, the cells were washed with cold PBS three times and fixed in 4% formaldehyde for 10 min at 4 °C. After permeabilization with 0.5% Triton X-100 for 10 min, cells were blocked by incubation with 4% bovine serum albumin (Sigma, MO,USA) in PBS for 30 min at room temperature, washed again, and incubated with 30 μg/mL human polyclonal anti-SARS-CoV-2 (CIGB, Cuba) for 2 h at room temperature.
Then, peroxidase-conjugated anti-rabbit secondary antibody 1:100 (Sigma, MO, USA) was incubated for 1 h at room temperature and washed 3 times with PBS. Finally, AEC substrate (Abcam, Cambridge, United Kingdom) was added and coverglass was mounted using 40% glycerol mounting medium and analyzed using a BX43 upright microscope (Olympus, USA).

Ramòn A.C., Perez G., Caballero E., Rosales M., Aguilar D., Vázquez‐Blomquist D., Ramos Y., Rodríguez A., Falcón V., Rodríguez M.P., Ke Y., Perera Y., & Perea S.E. (2021). Targeting of Protein Kinase CK2 Elicits Antiviral Activity on Bovine Coronavirus Infection.

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