The paraffin sections were deparaffinized in xylene, rehydrated in distilled water, treated with 3% H2O2 for 30 min at 37°C to block endogenous peroxidase activity and washed with 0.1M PBS 3 times. Nonspecific binding was blocked by 10% goat serum for 40 min at 37°C and was followed by incubation overnight at 4°C with antibodies against the following: DMT1 with IRE (#NRAMP21-A; 1:2,000) and DMT1 without IRE (#NRAMP23-A; 1:2,000) from Alpha Diagnostics International; Transferrin antibody (N3C3; 1:250) from GeneTex;TfR1 (1:100) from Thermo; FPN1 (1: 2,000; #MTP11-A) from Alpha Diagnostics International, Ceruloplasmin (1:50) from Origene; Hephaestin (1:200) from Alpha Diagnostics International; Ferritin(1:200) from Abcam; Hepcidin (1:50) from Abcam. On the following day, sections were washed in 0.1M PBS and incubated with biotinylated goat anti-rabbit IgG for 25 min. Finally, the sections were washed in 0.1M PBS, stained with DAB, dehydrated using graded ethanol, and cleared using xylene. Images of positive staining in the frontal cortex region were captured at 20× magnification by a Nikon 80i microscope and analyzed using ipwin32 software to measure Integral optical density (IOD).
Hepcidin
Manufactured by Abcam
Sourced in United Kingdom
Hepcidin is a peptide hormone that plays a key role in iron homeostasis. It is the main regulator of iron absorption and distribution in the body. Hepcidin acts by binding to the iron exporter ferroportin, causing its internalization and degradation, thereby reducing iron export from cells.
Automatically generated - may contain errors
Lab products found in correlation
Copper 2 chloride, by Merck Group (2 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (2 mentions)
Potassium chloride (kcl), by Merck Group (2 mentions)
Calcium chloride, by Merck Group (2 mentions)
Magnesium 2 chloride hexahydrate, by Avantor (2 mentions)
Ferrous ammonium sulfate, by Santa Cruz Biotechnology (2 mentions)
Nickel 2 sulfate, by Thermo Fisher Scientific (2 mentions)
Fe 2 bromide, by Merck Group (2 mentions)
Fe 3 sulfate hydrate, by Merck Group (2 mentions)
Buthionine sulphoximine bso, by Cayman Chemical (2 mentions)
β mercaptoethanol bme, by Bio-Rad (2 mentions)
Manganese 2 chloride, by Merck Group (2 mentions)
Copper 1 chloride, by Merck Group (2 mentions)
Cobalt 2 sulfate heptahydrate, by Merck Group (2 mentions)
N acetyl cysteine (nac), by Tokyo Chemical Industry (2 mentions)
Ascorbic acid, by Chem-Impex (2 mentions)
Ferritin, by Abcam (1 mentions)
Ecl detection kit, by Merck Group (1 mentions)
Nc membrane, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
10 protocols using hepcidin
1
Immunohistochemistry Analysis of Iron Regulatory Proteins
2
Metal Ion Supplementation Protocol
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Puromycin Dihydrochloride (>99%) was purchased from P212121. Zinc Sulfate was purchased as a 100 mM aq. Soln. from Alfa Aesar. Cobalt(II) sulfate heptahydrate (>99%) was purchased from Sigma-Aldrich: SIAL. Magnesium(II) chloride hexahydrate (ACS grade, >99%) was purchased from Amresco. Ferrous ammonium sulfate (99%) was purchased from Santa Cruz Biotechnology. Sodium chloride (99.5%) was purchased from Fisher Scientific. Nickel(II) sulfate (99.99%), Fe(II) bromide (98%), Fe(III) sulfate hydrate (97%), copper(II) chloride (99.999%), copper(I) chloride (>99.995%), calcium chloride (>97%), potassium chloride (>99%), and manganese(II) chloride (98%) were all purchased from Sigma Aldrich. N-acetyl cysteine (NAC) was purchased from TCI America (>98%). Ascorbic Acid was purchased from Chem Impex Int’l (99.8%). Buthionine sulphoximine (BSO) (>98%) was purchased from Cayman Chemical and β-mercaptoethanol (BME) (>98%) was purchased from BioRad. Hepcidin was purchased from abcam (1 mg/mL, 70–90%).
3
Western Blot Analysis of Iron Regulators
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Target cells were isolated in radioimmunoprecipitation assay (RIPA) lysis buffer (JRDun, Shanghai) and the extracted protein was collected by centrifuging. The same amount of protein was separated by SDS-PAGE gel and then transferred onto a nitrocellulose membrane (NC membrane, Millipore, USA). After blocking with 5% skim milk, the NC membrane was probed with relevant primary antibodies (hepcidin, DMT1, FPN1, CP, p-STAT3, STAT3, and GAPDH, Abcam, Cambridge), followed by appropriate secondary antibodies (HRP-labeled Donkey Anti-Goat IgG (H+L), HRP-labeled Goat Anti-Rabbit IgG (H+L) and HRP-labeled Goat Anti-Mouse IgG (H+L), Beyotime, China). Finally, the target bands were assessed using an ECL detection kit (Millipore, USA) and quantified by ImageJ software.
4
Immunohistochemical Analysis of Iron Homeostasis
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Samples were fixed in 4% paraformaldehyde in phosphate‐buffered saline (PBS) and then embedded in paraffin using standard procedures. Serial paraffin sections (4‐μm thickness) were prepared, and individual slides were stained with haematoxylin and eosin. Antigen retrieval was achieved by citrate buffer, pH 6.0. After antigen retrieval, immunohistochemical analyses were performed using following primary antibodies: Hepcidin (ab‐75883; AbCam, Cambridge, UK), L‐Ferritin (ab‐69090; AbCam), Ferroportin‐1 (sc‐49668; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), BMP‐6 (bs‐10090R; Bioss Antibodies, Bioss Antibodies, Woburn, MA) and Smad‐4 (sc‐7966; Santa Cruz Biotechnology, Inc.). Immunostainings were performed using the DakoCytomation Envision System (DAKO, Glostrup, Denmark) according to the manufacturer's instructions. Alexa Fluor‐conjugated secondary antibodies (Invitrogen, Carlsbad, CA) were used for immunofluorescent staining. The stained sections were examined with a stereomicroscope (MD5500D; Leica Microsystems, Wetzlar, Germany) and a confocal microscope (LSM700; Carl Zeiss, Jena, Germany).
5
Iron Homeostasis Protein Analysis
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Tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor (Thermo Fisher Scientific). Protein concentration in lysates was determined using BCA Protein Quantification Kit (Pierce) and as described previously (Sawicki et al., 2018 (link)). Equal amounts of proteins were resolved on 4–12% Novex Bis-Tris poly-acrylamide gel (Invitrogen) and blotted onto nitrocellulose membrane (Invitrogen). After blocking with tris-buffered saline containing 0.05% Tween 20 (Thermo Fisher Scientific) and 5% milk, the membrane was incubated overnight at 4°C in primary antibody against HPRT (ProteinTech), succinate dehydrogenase complex flavoprotein subunit A (SDHA, Invitrogen), FTN (Sigma-Aldrich), IRP2 (Novus Biologicals), TfR1 (Invitrogen), FPN1 (Novus Biologicals), Hepcidin (Abcam), Ubiquitin (Cell Signaling Technology), GAPDH (ProteinTech), and α-Tubulin (ProteinTech). The following day, membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (Jackson ImmunoResearch) for 1 hr and proteins were visualized using Super Surgical Western Pico ECL substrate (Pierce). Quantification of immunoblotting image was done using ImageJ (NIH).
6
Western Blot Analysis of Hepcidin
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After culturation as described above, the cells were washed with PBS twice, and protein was extracted using lysis buffer. Twenty micrograms of the total protein were loaded and electrophoresed on a 12% (w/w) sodium dodecyl sulfate–polyacrylamide gel after being quantified with a BCA kit (Beyotime, Shanghai, China). The resolved proteins were blotted onto a nitrocellulose membrane, and the membranes were then incubated with the primary antibody hepcidin (1:1000, Abcam, Cambridge, United Kingdom), followed by treatment with the corresponding secondary antibodies. The immunoreactive bands were visualized with NcmECL ultra-reagent (NCM Biotech, Suzhou, China). Densitometry of protein bands was performed using ImageJ (U.S. National Institutes of Health, Bethesda, MD, United States).
7
Ferroptosis Induction and Analysis
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Cell culture-tested ferric ammonium citrate (FAC) was purchased from Hi-Media. 5-Florouracil (5-FU), MTT, trypan blue, fetal bovine serum (FBS), RSL3, Ferrostatin (Ferro)-1, Deferoxamine (DFO), carbobenzoxy-valylalanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK), Bradford reagent for protein estimation, and streptomycin-penicillin solution were procured from Sigma-Aldrich (St. Louis, USA). Apoptosis detection annexin V and FITC kit, Propidium iodide (PI) staining kit for cell cycle analysis, and JC-1 staining for mitochondrial membrane potential detection kit were purchased from Abcam. Antibodies such as poly (ADPribose) polymerase 1 (PARP1), caspase-3, caspase-9, survivin, cyclin D1, p27, C-Myc, p21, Bcl-xL, Bak, Bax, and Bcl-2 were purchased from cell signal technology, whereas iron metabolism associated antibodies IRP-1, HMOX-1, TFRC, Hepcidin, and FTH1 were purchased from Abcam. Cell culture medium Roswell Park Memorial Institute (RPMI)-1640 was procured from Gibco Life Technologies (Waltham, USA).
8
Metal Ion Supplementation Protocol
9
Western Blot Analysis of Hepcidin and JAK-STAT Signaling
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Cells were lysed with RIPA lysis buffer. Tumor tissue samples were pelleted on ice. Protein lysates were subjected to western blot according to the method described previously [26 (link)]. All primary antibodies in the present study were listed as follows: hepcidin (Abcam, Cambridge, UK); STAT3, p-STAT3, JAK2, and p-JAK2 (CST, Beverly, USA); and β-actin (Santa Cruz, USA).
10
Quantifying Iron Regulatory Proteins
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Western blotting was performed as previously described. 27 The primary antibodies used in the experiment were as follows: TfR1, TfR2 and hepcidin (Abcam Inc., Cambridge, MA, USA).
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