Lipid peroxidation (LPO) is a major indicator of oxidative stress and cellular damage, frequently associated with environmental and toxicological stressors and mechanistically linked to ferroptotic regulated cell death (RCD). This protocol describes a simple and reproducible method for the qualitative in situ visualization of LPO in mosquito larvae using Schiff’s reagent, which histochemically labels reactive aldehyde groups [such as malondialdehyde (MDA)] generated during lipid degradation. Although Schiff’s reagent detects aldehydes commonly associated with lipid peroxidation, these compounds are not exclusive to LPO and may also arise from other oxidative processes. The method preserves tissue integrity, enabling direct, spatially resolved observation of oxidative damage in whole larvae. Following staining, larvae are rinsed in a stabilizing sulfite solution to maintain the characteristic magenta coloration. Using this assay, Culex quinquefasciatus larvae exposed to ferroptotic cyanobacteria, such as Synechocystis sp., exhibit a marked accumulation of lipid-derived aldehydes consistent with lipid ROS–mediated damage. This oxidative response is specifically suppressed by pre-treatment with the canonical ferroptosis inhibitor Ferrostatin-1 (Fer-1), which inhibits lipid peroxidation and significantly reduces larval mortality. As a complementary approach to traditional spectrophotometric assays such as thiobarbituric acid reactive substances (TBARS), this qualitative method enables in situ visualization of lipid peroxidation without tissue homogenization, providing a rapid and biologically informative screening tool for assessing ferroptosis-associated oxidative damage in Cx. quinquefasciatus and other biological models exposed to multiple stressors.

Key features

• Direct qualitative assessment: Provides visual, histochemical evidence of lipid peroxidation in situ, enabling rapid evaluation of oxidative membrane damage at the tissue and whole-organism levels.

• Preservation of spatial context: Allows localization of oxidative damage without tissue homogenization, maintaining tissue architecture and spatial resolution.

• Environmental and toxicological screening: Optimized for rapid detection of oxidative stress in mosquito larvae exposed to larvicides, biocontrol agents, and other environmental stressors.

• Complementary to quantitative assays: Functions as a screening tool that complements spectrophotometric methods such as thiobarbituric acid reactive substances (TBARS).

Oxidative stress

Lipid peroxidation

Mosquito larvae

Histochemistry

Environmental toxicology

Graphical overview

Schematic representation of the workflow used to detect lipid peroxidation–associated aldehydes in mosquito larvae by Schiff’s reaction. Larvae (10 per well) are distributed in a 24-well plate and washed in PBS, followed by permeabilization with PBS containing 0.1% Triton X-100 and subsequent PBS rinses. Schiff’s reagent is added where needed. The plate is covered with Parafilm, with holes in each well. Vacuum infiltration is applied in a desiccator covered with aluminum foil for 2 h to enhance reagent penetration. After staining, Schiff’s reagent is removed and replaced with sulfite solution to stabilize the coloration, followed by washing with distilled water to remove excess stain. Larvae are then mounted and photographed under a stereomicroscope. Reagent volumes (2 mL), incubation times, and light-protection steps are indicated at each stage. Plate layout: Row A, negative controls; A1–A3, stained with Schiff’s reagent; A4–A6, processed without Schiff’s reagent (unstained). Row B, positive controls exposed to 100 mM H₂O₂; B1–B3, stained; B4–B6, unstained. Row C, larvae exposed to live Synechocystis; C1–C3, stained; C4–C6, unstained. Row D, larvae exposed to ferroptotic Synechocystis; D1–D3, stained; D4–D6, unstained. Each experiment was performed in triplicate.