RT Journal Article
T1 Spatial Imaging and Quantification of Hydrogen Peroxide in Arabidopsis Roots: From Sample Preparation to Image Analysis
A1 Fenech, Mario
A1 Amorim-Silva, Vitor
K1 Arabidopsis - Aspectos moleculares
K1 Agua oxigenada
AB Reactive oxygen species (ROS) are central regulators of plant development and stress responses, with hydrogen peroxide (H2O2) acting as a key signaling molecule whose spatial distribution determines adaptive versus damaging outcomes. Accurate detection of H2O2 at tissue and cellular resolution is therefore essential for understanding redox-dependent regulation of plant growth. A variety of techniques have been used to monitor H2O2, including bulk spectrophotometric and fluorometric assays, genetically encoded sensors for real-time measurements, and chemical probes for in situ detection. While these approaches differ in sensitivity, specificity, and temporal resolution, many are limited by a lack of spatial information, technical complexity, or dependence on transgenic material. Here, we present a detailed protocol for 3,3′-diaminobenzidine (DAB)-based histochemical detection of H2O2 in seedling roots, covering staining, imaging, and semi-quantitative image analysis using open-source software (FIJI/ImageJ). The method relies on peroxidase-mediated oxidation of DAB, resulting in a stable, light-resistant, and insoluble precipitate that enables visualization of H2O2 accumulation with high spatial resolution. This protocol provides a robust, accessible, and genetically independent approach for spatial analysis of H2O2 in plant tissues. Its simplicity, compatibility with diverse genotypes and treatments, and suitability for semi-quantitative analysis make it a valuable tool for examining the spatial distribution of H2O2, thereby providing spatial insight into redox-related regulatory processes during plant development and stress responses.
PB Bio-Protocol
SN 2331-8325
YR 2026
FD 2026-04-20
LK https://hdl.handle.net/10630/46442
UL https://hdl.handle.net/10630/46442
LA eng
NO Fenech, M. and Amorim-Silva, V. (2026). Spatial Imaging and Quantification of Hydrogen Peroxide in Arabidopsis Roots: From Sample Preparation to Image Analysis. Bio-protocol 16(8): e5659. DOI: 10.21769/BioProtoc.5659
NO Programa Emergia 2023 de la Consejería de Universidad, Investigación e Innovación de la Junta de Andalucía
DS RIUMA. Repositorio Institucional de la Universidad de Málaga
RD 4 may 2026