Antibodies against nuclear proteins, as well as isotype-matched control IgGs, were preconjugated to Allophycocyanin (APC) using lightning-link APC conjugation kit (Innova Biosciences). The monoclonal antibody against NeuN coupled to Alexa-Fluor 488 (Millipore, MAB 477X) was used for neuron-specific labeling. The antibodies and reagents used in this study are listed in Supplementary Table 3 . Approximately 1–2 μg of antibody was added to1×106 nuclei/ml and incubated at 4°C for 12 hrs with gentle rocking in the dark. Labeled nuclei were washed twice by centrifugation and filtered to remove clumps before performing FACS. The DNA binding dyes DAPI or propidium iodide were used for gating of nuclei. Aggregate discrimination was achieved based on the height, area and width parameters for front scattering (FSC) and side scattering (SSC). The purity and identity of isolated neuronal nuclei was further confirmed by microscopy and epigenetic analysis. For each antibody, the geometric mean value of single-nuclei events that is above the matched nonspecific IgG control was determined. An average of 10,000 events was acquired for FACS analysis. For ChIP assays, ~1×106 FACS-sorted neuronal nuclei were collected by centrifugation.
Mab 477x
Manufactured by Merck Group
The MAB 477X is a high-performance laboratory instrument designed for researchers and scientists. It is a versatile and reliable tool that offers precise and efficient analysis capabilities. The core function of the MAB 477X is to facilitate the measurement and characterization of samples in a wide range of applications. However, a detailed description of its intended use cannot be provided while maintaining an unbiased and factual approach.
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Lab products found in correlation
2 protocols using mab 477x
1
Isolation and Characterization of Neuronal Nuclei
2
Isolation and Characterization of Neuronal Nuclei
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Antibodies against nuclear proteins, as well as isotype-matched control IgGs, were preconjugated to Allophycocyanin (APC) using lightning-link APC conjugation kit (Innova Biosciences). The monoclonal antibody against NeuN coupled to Alexa-Fluor 488 (Millipore, MAB 477X) was used for neuron-specific labeling. The antibodies and reagents used in this study are listed in Supplementary Table 3 . Approximately 1–2 μg of antibody was added to1×106 nuclei/ml and incubated at 4°C for 12 hrs with gentle rocking in the dark. Labeled nuclei were washed twice by centrifugation and filtered to remove clumps before performing FACS. The DNA binding dyes DAPI or propidium iodide were used for gating of nuclei. Aggregate discrimination was achieved based on the height, area and width parameters for front scattering (FSC) and side scattering (SSC). The purity and identity of isolated neuronal nuclei was further confirmed by microscopy and epigenetic analysis. For each antibody, the geometric mean value of single-nuclei events that is above the matched nonspecific IgG control was determined. An average of 10,000 events was acquired for FACS analysis. For ChIP assays, ~1×106 FACS-sorted neuronal nuclei were collected by centrifugation.
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