Numbers of macrophages and fibroblasts were quantified by immunofluorescence in fixed mid-LV sections. Sections were cleared and rehydrated in decreasing concentrations of ethanol. Immunofluorescence was performed using the Opal 7-color Automation Kit (Perkin Elmer #NEL80100KT; Boston, MA) according to the manufacturer’s instructions. Samples were incubated with primary antibodies against alpha-smooth muscle actin (α-SMA; 1:1000; Sigma Aldrich #2547), inducible nitric oxide synthase (iNOS; 1:100; Novus Biologicals #NB300), or CD31 (1:100; Cell Signaling Technologies #77699) followed by HRP-conjugated secondary antibody supplied by the kit, and finally conjugated to Opal 520 fluorophore and counterstained with DAPI. At least 10 random fields per sample were obtained at 40X using the Mantra System (Perkin Elmer; Boston MA) with the observer blinded to the sample, and cells counts were automated using inForm software (Perkin Elmer).
Opal 7 color automation kit
Manufactured by PerkinElmer
Sourced in United States
The Opal 7-color Automation Kit is a laboratory equipment product designed for multiplexed immunofluorescence analysis. It provides a standardized workflow for simultaneous detection and quantification of up to 7 protein targets within a single tissue sample.
Automatically generated - may contain errors
2 protocols using opal 7 color automation kit
1
Quantifying Cardiac Cell Populations
2
Multiparametric Immunohistochemical Analysis
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LV mid-sections were fixed in 10% zinc-buffered formalin, paraffin-embedded, and sectioned at 5 µm. Slides were deparaffinized and rehydrated. Immunohistochemical staining was performed using the Opal 7-color Automation Kit (Perkin Elmer #NEL80100KT; Boston, MA, USA). Sections were stained with the following primary antibodies conjugated to Opal fluorophores: PDGFRα (1:100, R&D Systems; #AF1062; Minneapolis, MN) conjugated to Opal 520, and CX3CL1 (1:500; R&D #MAB571), CCL5/RANTES (R&D #AF478), VEGF (Abcam #ab51745), or thrombospondin 1 (Thbs1; 1:100; R&D Systems #AF3074) conjugated to Opal 650. Nuclei were counterstained with DAPI. Opal 520 and 650 fluorophores were visualized with FITC and Cy5 channels, respectively. Images were obtained using the Mantra System (Perkin Elmer) and quantified using inForm software (Perkin Elmer).
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