Ab238538

MDA and 4-HNE, the main products of lipid peroxidation, were measured by their detection kits (S0131M, Beyotime, and ab238538, Abcam) (Rong et al., 2021a (link)). Briefly, for MDA detection, the samples were mixed with the working solution pre-prepared, then heated at 100°C for 15 min and cooled to room temperature. The absorbance at 532 nm was measured under a microplate reader (BioTek). For 4-HNE detection, after adding 50 μl of the diluted anti-4-HNE antibody and 100 μl diluted secondary antibody to samples, respectively, and incubating for 1 h, we washed the mixture and stopped the reaction. The absorbance at 450 nm was detected on a microplate reader (BioTek). The relative GSH level was tested by the GSH assay kit (S0053, Beyotime). Analogously, the sample was mixed with the GSH working solution, and the absorbance at 405 nm was measured on a microplate reader (BioTek). The relative Fe²⁺ concentration was detected by an iron assay kit (ab83366, Abcam). For iron detection, 5 μl of assay buffer was added to each sample and then incubated at 37°C for 30 min, followed by adding 100 μl iron probe to the mixture and incubated at 37°C for 60 min in the dark. Finally, the absorbance at 593 nm was measured on a microplate reader (BioTek).

To ascertain the involvement of lipid peroxidation in lung tumorigenesis, the cells were stained by 5 μM fluorescent probe C11-BODIPY^581/591 for 30 min at 37 °C followed by flow cytometry. To visualize the membrane, tissue slides were incubated with 25 μg/ml Concanavalin A-Alexa Fluor^TM 350 (Thermo, #C11254) following 5 μM C11-BODIPY^581/591 staining at 37 °C for 30 min. Images were captured at emission at 580/600 nm (the non-oxidized form, red) and 490/510 nm (the oxidized form, green) and then merged to demonstrate the fraction of the oxidized C11-BODIPY^581/591. The malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) concentration were assessed using lipid peroxidation assay kits (Abcam, #ab118970 and #ab238538). IHC was measured to detect the expression level of 4-HNE using anti-4-HNE antibodies (Abcam, #ab46545).

According to the previous grouping, the brain homogenates of mice 3 days after ICH were assessed using ELISA. IL-1β (p1301, Biotime, Shanghai, China), IL-6 (PI326), TNF-α (PT512), and 4-HNE (ab238538, Abcam) levels were determined according to the manufacturers’ instructions. The concentrations of cytokines and 4-HNE (pg/mL) were determined using standard curves obtained from known amounts of IL-1β, IL-6, or TNF-α and were expressed as a percentage relative to their concentrations in the ICH group. BV2 microglia were divided into groups according to the previous experimental design section, the corresponding reagents were added to the cell culture media, and the cells were cultured for 24 hours. At the end of the incubation, the culture media were collected. The concentrations of cytokines and 4-HNE (pg/mL) in the culture media were expressed as a percentage relative to their concentrations in the culture media of cells stimulated with hemin (10 μM).

Mouse RPE/choroid cups were sonicated in RIPA buffer (50 mM Tri-Cl pH 7.4, 150 mM NaCl, 0.5% NP40, 0.5% sodium deoxycholate, 1 mM EGTA, 5% Glycerol) plus protease inhibitor/phenylmethylsulfonyl fluoride/phosphatase inhibitor). The homogenates were centrifuged for 15 min at 12,000 × g at 4 °C. Protein concentration was determined by BCA assay (Bio-Rad Laboratories) and 16 μg homogenates (in 50 μl) were used in each 96-well for 4-HNE concentration assay, as described^{81 (link)}. 4-HNE concentration was quantified using an ELISA kit following the manufacturer’s protocol (#Ab238538, Abcam). The ELISA data analysis was collected by SPECTRA Max (450 nm) M2e plate reader (Molecular Devices Corp) and processed by SoftMax Pro 5.4. The average (presented as μg per ml) and SEM were calculated based on the 4-HNE-BSA standard curves.