Ab183781

The following antibodies were used: β-actin antibody (cw0096m, CWbio, China), FPN1 antibody (MTP11-S, ADI, USA), DMT1 (+IRE) antibody (NRAMP21-S, ADI, USA), 4-HNE antibody (HNE-11S, ADI, USA), TfR1 antibody (ab84036, Abcam, USA), L-ferritin antibody (ab109373, Abcam, USA), H-ferritin antibody (ab183781, Abcam, USA), hepcidin antibody (ab30760, Abcam, USA), Aβ_22–35 antibody (A3356, Sigma, USA), phospho-p38 (p-p38) antibody (4511S, CST, USA), p38 antibody (8690S, CST, USA), phospho-ERK (p-ERK) antibody (9102S, CST, USA), ERK antibody (4372S, CST, USA), Bcl-2 antibody (12789-1-AP, Proteintech, China), Bax antibody (50599-2-Ig, Proteintech, China), GFAP antibody (MAB360, Millipore, USA), Iba1 antibody (MABN92, Millipore, USA), NeuN (ab104224, Abcam, USA), CD31 (77699T, CST, USA), PSD-95 (ab18258, Abcam, USA), anti-rabbit IgG (RPN4301, Amersham, UK), anti-mouse IgG (RPN4201, Amersham, UK), DyLight 488 goat anti-mouse IgG (A23210, Abbkine, USA), DyLight 549 goat anti-rabbit IgG (A23320, Abbkine, USA), and DyLight 549 goat anti-mouse IgG (A23310, Abbkine, USA).

Protein samples were collected from tissues or cells with RIPA Lysis Buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS). Protein was loaded onto SDS-PAGE gel electrophoresis system (PowerPac, Bio-Rad, 164-5052). PVDF membranes were first used for transfer prior to membrane blocking with 5–8% skimmed milk for 1 h. They were then incubated with primary antibodies for 8–12 h (4°C), and were finally incubated with secondary HRP-conjugated antibodies (anti-mouse, Proteintech #SA00001-1, 1/10,000; anti-rabbit, Proteintech #SA00001-2, 1/10,000). WB densitometry was quantified using the Image Lab system. Antibodies used included anti-Adipsin rabbit monoclonal antibody (Abcam, ab213177, 1/1,000), anti-transferrin rabbit monoclonal antibody (Abcam, ab109503, 1/8,000), HRP anti-beta actin mouse monoclonal antibody (Abcam, ab20272, 1/5,000), anti-TFRC rabbit polyclonal antibody (Proteintech, 10084-2-AP, 1/2,000), anti-ferritin heavy chain rabbit monoclonal antibody (Abcam, ab183781, 1/1,000), anti-COX2 rabbit polyclonal antibody (ABclonal, A1253, 1/3,000), anti-GPX4 rabbit monoclonal antibody (ABclonal, A11243, 1/1,000), anti-GAPDH rabbit polyclonal antibody (Proteintech, 10494-1-AP, 1/8,000).

Once sacrificed, rats underwent decapitation, the cerebrum was extracted and tissue from peri-haemorrhagic areas was harvested from the various cohorts. Following embedding in paraffin, the specimens were sliced into 5-µm sections; xylene was utilised for dewaxing. The sections were then soaked in antigen repair solution and a low flame was applied for 10 min. After cooling to the ambient temperature, the samples underwent incubation in 3% hydrogen peroxide; goat serum was used for blocking. Threefold 5-min washing in phosphate-buffered saline was performed at each stage of the process.
The sections then underwent overnight incubation at a temperature of 4 °C with primary antibodies, i.e. rabbit anti-Nrf2 (1:200, no. bs-1074R, Bioss), rabbit anti-FTH1 (1:400, no. ab183781, Abcam) and rabbit anti-GPX4 (1:300, no. ab125066, Abcam). Incubation for 30 min at 37 °C with the equivalent secondary antibody, i.e. biotinylated goat anti-rabbit IgG (1:200, no. A0277, Beyotime) was performed. Lastly, labelling with horseradish, produced in DAB, was undertaken. The samples were soaked in distilled water and then haematoxylin counterstaining was conducted, followed by desiccation and sealing of the sections. A microscope (DP73, Olympus Corporation, Tokyo, Japan) was employed to acquire images at × 400 magnification.