To validate the in vivo MRI imaging, equivalent sections of the scanned aortas were harvested and embedded in OCT. Serial 4-μm–thick cryostat sections were stained either with oil red for lipid deposits detection and characterization with the following antibodies: CD163 and CD68 (macrophages), and α-actin (smooth muscle cells). The secondary antibodies and ABComplex/HRP were added and sections were stained with 3, 30-diaminobenzidine, counterstained with hematoxylin and mounted in DPX. For CD163 detection Tyramide Signal Amplification Biotin Kit (PerkinElmer, NEL700A001KT) was needed to improve signal detection. Images were taken using a Nikon Eclipse E400 microscope and Nikon ACT-1 software.
Tyramide signal amplification biotin kit
Manufactured by PerkinElmer
The Tyramide Signal Amplification Biotin Kit is a laboratory tool used to enhance the detection and visualization of target molecules in biological samples. It provides a method for amplifying signal intensity, enabling more sensitive and reliable detection of low-abundance targets.
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Lab products found in correlation
Act 1, by Nikon (1 mentions)
Eclipse e400 microscope, by Nikon (1 mentions)
Ec plan neofluar objective, by Zeiss (1 mentions)
Axiocam hrm camera, by Zeiss (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Axio imager z1 fluorescent microscope, by Zeiss (1 mentions)
B220 fitc ra3 6b2, by Thermo Fisher Scientific (1 mentions)
2 protocols using tyramide signal amplification biotin kit
1
Histological Validation of Aortic MRI
2
Visualization of Extrafollicular Plasma Cells
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Frozen spleen sections (6 μm) were fixed in ice-cold acetone and subsequently blocked with 0.2% BSA. Streptavidin and biotin block (Vector Laboratories) were used as recommended by manufacturer's protocol. For visualization of extrafollicular responses, 0.3% H 2 O 2 was used to quench endogenous peroxidase activity. Anti-mouse CD138-biotin was used with Tyramide Signal Amplification-biotin kit (Perkin Elmer) as recommended by manufacturer's protocol to amplify signal. To detect CD138 + cells, streptavidin-Dylight 549 (Jackson ImmunoResearch) was used. DCs were stained with Armenian hamster anti-mouse CD11c (HL3; BD Pharmingen) and detected with anti-Armenian hamster Dylight 649 (Jackson ImmunoResearch), while B cells were detected with B220-FITC (RA3-6B2, eBioscience). To detect IgM + plasmablasts, IgM-FITC (1B4B1, Southern Biotech) was used. Slides were mounted with fluorescent mounting medium (DAKO). Images were acquired using Axio Imager.Z1 fluorescent microscope (Zeiss) equipped with EC Plan-NEOFLUAR objective lenses (10×/0.3 and 40×/0.75), AxioCam Hrm camera (Zeiss), and AxioVision software v4.8.2.0.
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