High‐Content Immunofluorescence Quantification of Transcription Factors: Application to pSMADs for BMP/TGFβ Signaling Pathways

High-throughput immunofluorescence (IF) detection has become an essential tool in drug discovery for analyzing multiple markers and providing insights into cellular pathways. While IF techniques have been established for decades, high-content IF protocols vary among laboratories, highlighting the need for standardized and quantitative workflows. In addition, recent technological advances allow single-cell IF detection, which often offers a detailed perspective on cell population distribution within the sample. This protocol presents an optimized IF method for detecting and quantifying nuclear pSMAD signaling in adherent cells cultured on biomaterial substrates. Notable optimizations include fixation buffers, antibody concentrations, and an automated image analysis workflow. This process significantly reduces assay time, enables multiplex biomarker detection, and describes a method for overcoming the optical artifact observed with biomaterials. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescence staining of nuclear pSMAD signaling on biomaterials and on glass substrates Basic Protocol 2: High-content imaging and single-cell quantification of SMAD pathway Support Protocol 1: Preparation of 3.7% PFA (500 ml) Support Protocol 2: Macro in ImageJ/Fiji (version 1.54p).