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Inside this application protocol
Review a step-by-step workflow for validating ICC antibody specificity using iPSC mRNA transfection, quantitative CQ1 imaging, and automated CellPathfinder analysis to classify antibodies before use.
- iPSCs seeded at 25,000 cells/well in 96-well plates were transfected with ALDH1L1 or GFAP mRNA; untransfected wells served as negative controls, with samples prepared in triplicate.
- Automated CQ1 confocal imaging and CellPathfinder segmentation counted more than 10,000 cells per well, with fluorescence intensity histograms used to set thresholds and classify antibodies as Good, Fair, or Bad based on a 5% positive-cell cutoff in negative controls.
- Validated antibodies were applied to Quick-Glia Astrocytes fixed at day 43, yielding 50% GFAP-positive and 94% ALDH1L1-positive cells, with thresholds adjusted for the ALDH1L1 antibody's observed non-specific background signal.
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