Spatial Imaging and Quantification of Hydrogen Peroxide in Arabidopsis Roots: From Sample Preparation to Image Analysis.

Abstract

Reactive oxygen species (ROS) are central regulators of plant development and stress responses, with hydrogen peroxide (H 2 O 2 ) acting as a key signaling molecule whose spatial distribution determines adaptive versus damaging outcomes. Accurate detection of H 2 O 2 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 H 2 O 2 , 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 H 2 O 2 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 H 2 O 2 accumulation with high spatial resolution. This protocol provides a robust, accessible, and genetically independent approach for spatial analysis of H 2 O 2 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 H 2 O 2 , thereby providing spatial insight into redox-related regulatory processes during plant development and stress responses. Key features • Built upon methods developed by Thordal-Christensen et al. [1] and Daudi and O'Brien [2], with a specific focus on root staining. • Includes a downstream image analysis pipeline for semi-quantitative H 2 O 2 measurement in DAB-stained roots using the open-source software FIJI/ImageJ. • Provides detailed, step-by-step video tutorials for image analysis in FIJI/ImageJ. • Includes a Fiji/ImageJ script (Macro 1) for automating the application of fixed-intensity scaling using Spectrum LUT.

Plant phenotyping relevance

植物組織内の過酸化水素を画像取得・画像解析で空間的かつ半定量的に測定する実験プロトコルが中心であり、植物の生理状態を抽出するフェノタイピング手法に該当する。

Code and data availability

This is a Bio-protocol methods article describing DAB staining of Arabidopsis roots and FIJI/ImageJ-based quantification. The only paper-specific analysis assets mentioned are a Fiji/ImageJ macro (Macro 1) said to be in the supplementary information and an 'in-house ad-hoc' R script; neither is given a public deposit/`

No evidence-backed public reproduction asset is currently recorded.