Ab65080

A polyclonal rabbit anti-hepcidin 25 (Abcam, ab30760), recognising a 2.8 kDa protein [53 (link)], monoclonal antibody (MAB) human anti-ferritin light chain (Abcam, ab69090), human anti-ferritin heavy chain (Abcam, ab65080), anti-DMT1 (Abcam, ab123085), anti-FPN (Abcam, ab85370), anti-CD42b (Abcam, ab104704), anti-rabbit polyclonal glycophorin (Abcam, ab196568), anti-GFAP (Abcam, ab48050), anti-CD68 (Sigma-Aldrich, AMAB98073), MAB anti-CD11b (Thermo Fisher, Waltham, MA, USA, mAbM1/70), GFAP (Abcam, ab48050), MAB anti-Iba1 (Thermo Fisher, MAB M1/70), polyclonal anti-Iba1 (Wako, Fujifilm, Tokyo, Japan, catalogue number 019-19741), MAB anti-IL-6 (Thermo Fisher, catalog number M620), MAB IL-1β (Thermo Fisher, ILB1-H67), MAB anti-β amyloid 42 (Covance, Princeton, NJ, USA, SIG 39320), anti-phospho-tau (AT8, Thermo Fisher, MN1020), SOD1 (Abcam, ab252426), S100β (Abcam, ab218514), RUNX1 (Sigma-Aldrich, HPA004176) and OLIG2 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-365644) were used for IHC or Western blotting. The following secondary antibodies were used: biotinylated goat anti-rabbit-Ig and biotinylated horse anti-mouse (both from Vector Laboratories, 1:250 for IHC), Alexa Fluor 568-labelled donkey anti-mouse-Ig, Alexa Fluor 488-labelled donkey anti-rabbit-Ig and Alexa Fluor 568-labelled donkey anti-goat-Ig (all from Invitrogen, 1:1000 for immunofluoresence).

A polyclonal rabbit anti-hepcidin 25 (Abcam ab30760) recognising a 2.8 kDa protein (Raha-Chowdhury et al., 2015 (link)), human anti-ferritin light chain (Abcam ab69090), human anti-ferritin heavy chain (Abcam ab65080), Anti-FPN (Abcam ab85370), anti-GFAP (Abcam ab48050), anti-CD68 (Sigma–Aldrich, MAB98073), and monoclonal anti-Iba1 (Thermo Fisher Scientific, MAB M1/70), Polyclonal anti-Iba1(Wako cat number 019-19741), monoclonal anti-IL-6 (Thermo Fisher, cat number M620), monoclonal anti-IL-1β (Thermo Fisher Scientific, cat number ILB1-H67), Anti-β amyloid (Covance cat number SIG 39320), Anti-Phospho-Tau (AT8, Thermo Fisher Scientific, cat number MN1020), was used for IHC. The following secondary antibodies were used: biotinylated goat anti-rabbit-Ig and biotinylated horse anti-mouse (both from Vector Laboratories, 1:250 for IHC); Alexa Fluor 568-labeled donkey anti-mouse-Ig, Alexa Fluor 488-labeled donkey anti-rabbit-Ig, and Alexa Fluor 568-labeled donkey anti-goat-Ig (all from Invitrogen, 1:1,000 for immunofluorescence).

Three μm paraffin sections were stained on a Dako Autostainer Universal Staining System (Dako, Glostrup, Denmark). The sections were deparaffinized, and heat induced epitope retrieval (HIER) was performed by incubation in a buffer solution consisting of 10 mmol/L Tris-base and 0.5 mmol/L EGTA, pH 9. After blocking of endogenous peroxidase activity by incubation in 1.5% hydrogen peroxide, the sections were incubated for 60 min with primary antibodies against TfR1 (CD71) (10F11, 1+50, NovoCastra, Newcastle, United Kingdom), FTH (ab65080, 1+800, Abcam, Cambridge, United Kingdom), or FTL (ab69090, 1+1000, Abcam). The antigen-antibody complex was detected using EnVision (Dako). Visualization was performed using DAB (diaminobenzidine) as chromogen. Finally, sections were counterstained with Mayer’s hematoxylin, and coverslips were mounted with Aquatex. Paraffin sections from a TMA containing 28 normal tissues and 12 cancers were used as controls. Omitting primary antibodies served as negative controls as well as controls for non-specific staining related to the detection system.

Dihydroartemisinin (DHA, D140839, purity: ≥98%), hemin (H140872) and chloroquine (CQ, C193834) were purchased from Aladdin (China). Dimethyl sulfoxide (DMSO, A503039) and ferric ammonium citrate (FAC, A500061) were got from Sangon Biotech (China). Deferoxamine (DFO, D9533) and protoporphyrin IX zinc(II) (ZnPPIX, 282820) were purchased from Sigma-Aldrich (United States). Z-VAD-FMK (HY-16658B), ferrostatin-1 (Fer-1, HY-100579) and doxorubicin hydrochloride (DOX, HY-15142) were purchased from MedChemExpress (United States). And the antibody to ACSL4 (ab155282), GPX4 (ab125066), xCT (ab175186), HO-1 (ab82585), TfR1 (ab84036), FTH1 (ab65080) and NCOA4 (ab86707) were all purchased from Abcam (United Kingdom). Anti-β-actin and goat anti-rabbit IgG H&L were purchased from Bioker (China).

Total protein was extracted by RIPA lysis buffer (P0013, Beyotime) from cells. The concentration of the total protein was detected by the BCA kit (Bio-Rad, Hercules, CA, USA). Approximately 20 μg protein was separated by 12% SDS-PAGE and then transferred onto a PVDF membrane followed by blocking with 5% skimmed milk at 25 °C for 2 h. After that, PVDF membrane was incubated with primary antibodies overnight at 4 °C. Primary antibodies: HBx (1:1000, ab2741, Abcam), PRMT9 (1:1000, PA5-48942, Invitrogen), FTH1 (1: 1000, ab65080, Abcam), GPX4 (1:1000, sc-166570, Santa Cruz Biotechnology), 4-HNE (1: 1000, ab46545, Abcam), HSPA8 (1: 4000, 10654-1-AP, Proteintech), MMA (1:1000, 8015S, CST), sDMA (1:1000, 13222S, CST), GAPDH (1:2000, 60004-1-Lg, Proteintech). Next, the PVDF membranes were incubated with secondary antibodies: Goat Anti-Mouse IgG H&L(HRP) (1: 1000, ab205719, Abcam) and Goat Anti-Rabbit IgG H&L(HRP) (1: 20000, ab6721, Abcam) for 2 h at 25 °C. The bands were exposed to ECL reagent and photographed by Chemiluminometer (Clinx Science Instruments, Shanghai, China) and quantified by Image J software.

Proteins were extracted from the neuron and the peri-hematoma cortex tissue as described previously [24 ]. Immunoblot analyses were performed using the following primary antibodies: cleaved-caspase3 (1:500; ab49822; abcam), GP4X (1:750; 14432-1-AP; proteintech), FTH1 (1 μg/ml; ab65080; abcam), ACSL-4 (1:100; 22401-1-AP; proteintech), SLC7A11 (1:1000; 26864-1-AP; proteintech), occludin (1:1000; 27260-1-AP; proteintech), claudin-5 (1:5000; ab131259; abcam), ZO-1 (1:5000; 21773-1-AP; proteintech), β-actin (1:5000; 66009-1-Ig; proteintech) at 4 °C overnight. Secondary antibodies included anti-rabbit IgG and anti-mouse IgG (1: 5000; SA00001-1; SA00001-2; proteintech). The proteins were visualized using the ChemiDoc XRS imaging system.