Immunocytochemistry was conducted as previously described (Pediaditakis et al., 2021 (link)). Cells were blocked on the Brain-Chip in phosphate-buffered saline (PBS) containing 10% donkey serum (Sigma) at 4°C overnight. Saponin 1% was used to permeabilize membrane when required. Primary antibodies were MAP2 (Thermo Fisher Scientific; MA512826), VGLUT1 (Thermo Fisher Scientific; 48-2400), Synaptophysin (Abcam; 32127), GFAP (Abcam; ab53554), GLAST (Invitrogen; PA5-19709), s100β (Abcam; 52642), NG2 (Abcam; ab83178), αSMA (Abcam; 7817), IBA1 (FUJIFILM; 019-19741), CD68 (Abcam; ab213363), ICAM-1 (R&D Systems; BBA3), Ki67 (Abcam; 197234), ZO-1 (Thermo Fisher Scientific; 402200), Occludin (Invitrogen; OC-3F10), Claudin-4 (Invitrogen; 329494), TRPV6 (Proteintech;13411-1-AP), PECAM1 (Thermo Fisher Scientific; RB-1033-P1), CD11b (Invitrogen; MA1-80091), CD45 (Invitrogen; 17-0409-42), GLUT1 (Thermo Fisher Scientific; SPM498), P-gp (Thermo Fisher Scientific; MA5-13854), MRP-1 (Millipore; MAB4100), Transferrin receptor (Abcam; 216665). Chips treated with corresponding Alexa Fluor secondary antibodies (Abcam) were incubated in the dark for 2 h at room temperature. Cells were then counterstained with nuclear dye DAPI. Images were acquired with an inverted laser-scanning confocal microscope (Zeiss LSM 880).
Transferrin receptor
Manufactured by Abcam
Sourced in United Kingdom
Transferrin receptor is a protein that binds and internalizes transferrin, which is a major iron-binding protein in the blood. It is involved in the regulation of cellular iron uptake and is expressed on the surface of many cell types.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (2 mentions)
Ferritin, by Abcam (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Synaptophysin, by Abcam (1 mentions)
Occludin, by Thermo Fisher Scientific (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Alexa fluor secondary antibodies, by Abcam (1 mentions)
Vglut1, by Thermo Fisher Scientific (1 mentions)
Pecam 1, by Thermo Fisher Scientific (1 mentions)
Claudin 4, by Thermo Fisher Scientific (1 mentions)
α sma, by Abcam (1 mentions)
Glut1, by Thermo Fisher Scientific (1 mentions)
Cd11b, by Thermo Fisher Scientific (1 mentions)
Trpv6, by Proteintech (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Icam 1, by R&D Systems (1 mentions)
Iba 1, by Fujifilm (1 mentions)
Ferroportin, by Novus Biologicals (1 mentions)
Vegfa, by Abcam (1 mentions)
Tgf β, by Affinity Biosciences (1 mentions)
9 protocols using transferrin receptor
1
Comprehensive Immunocytochemistry on Brain-Chip
2
Western Blot Analysis of Mouse Heart Proteins
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Mouse heart tissue/cells were lysed for 30 min with RIPA buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors (Beyotime, China) on ice. The lysate was centrifuged at 12 000 rpm for 15 min at 4 °C, and the protein concentrations of the supernatant were analyzed with a BCA kit (Beyotime, China). Each sample (20 µg of protein) was separated by SDS‒PAGE gels and transferred to PVDF membranes. The membranes were immersed in 5% milk in TBST buffer for 1 h at room temperature, followed by incubation with primary antibodies against β‐actin (CST, 1:1000), bax (Abcam, 1:1000), caspase3 (CST, 1:1000), caspase 8 (CST, 1:1000), Cyt‐c (Proteintech, 1:1000), GPx‐4 (Abcam, 1:1000), Bcl‐2 (Affinity, 1:1000), TGF‐β (CST, 1:1000), COX‐2 (Affinity, 1:1000), VEGF A (CST, 1:1000), FAK (CST, 1:1000), ERK1/2 (CST, 1:1000), p38 (CST, 1:1000), transferrin receptor (Abcam, 1:1000), and ferroportin (Novus, 1:1000) at 4 °C overnight. Then, the membranes were washed three times with TBST, followed by incubation with secondary antibodies (1:10 000) for 1 h at room temperature. The bands were visualized by using a gel documentation system (Bio‐Rad, USA) and quantified by ImageJ software.
3
Western Blot Analysis of ABC Transporters
4
Iron Chelation and Neuroprotection
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3,4-Dihydroxyhydrocinnamic acid was purchased from HEOWNS (China, Tianjin). Spermine (SPM) was purchased from Aladdin (China, Shanghai). MTT, 6-hydroxydopamine hydrochloride (6-OHDA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Calcein AM was purchased from Abcam (Cambridge, UK). Deferoxamine mesylate (>98%) was purchased from MedChemexpress (New Jersey, USA). LDH Cytotoxicity Assay Kit, Annexin V-FITC Apoptosis Detection Kit, Reactive Oxygen Species Assay Kit, Lipid Peroxidation MDA Assay Kit, Total Superoxide Dismutase Assay Kit with WST-8, JC-1staining Kit were purchased from Beyotime (Nanjing, China). Hoechst 33,342 was purchased from Invitrogen (Carlsbad, CA, USA). Rabbit antibodies to Bcl-2, Bax, cytochrome C, Transferrin Receptor, Ferritin were purchased from Abcam (Cambridge, UK). Tyrosine hydroxylase (TH) and α-synuclein were purchased from Cell Signaling Technology (Beverly, MA, USA). Caspase-3, Caspase-9, PARP were purchased from Wanleibio. (China, Beijing). Ferroportin1 was purchased from Novus Biologicals (SanDiego, California, USA). β-Actin, α-tublin, hephaestin, DMT1 and HRP-conjugated goat anti-rabbit (mouse) IgG were purchased from Proteintech. (Rosemont, USA). Other chemicals and regents available were purchased form local commercial sources. PC12 cell line was gifted by professor Lu Ke at China Medical University.
5
Protein Expression Analysis in Cells
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Cells were washed with cold PBS, scraped, and lysed with 5% SDS/125 mM Tris-HCl (pH 6.8) at 99 °C. Protein levels were assessed by Western blotting. Protein concentrations in the samples were determined using the BCA™ protein assay kit (Pierce). Proteins were separated by 12% SDS-PAGE, and then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked and incubated overnight at 4 °C with the following primary antibodies: ferritin (Abcam, 1:1000), transferrin receptor (Abcam, Cambridge, UK, 1:1000), LC3B (Cell Signaling, 1:1000), PARP (Cell Signaling, 1:1000), PI3K (Cell Signaling, 1:1000), Akt (Cell Signaling, 1:1000), phospho-Akt (Cell Signaling, 1:1000), phospho-H2A.X (Ser139) (Cell Signaling, 1:000), and survivin (Cell Signaling, 1:1000). Membranes were washed and incubated with anti-rabbit and anti-mouse ECL antibodies (GE Healthcare) for 1 h at room temperature and washed again. After antibody incubation, band intensity was detected via chemiluminescence and imaged using an Azure Biosystems c600.
6
Antibody-Functionalized Liposome ELISA
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The functional activity of the antibodies conjugated to the liposomes was analyzed by the enzyme-linked immunosorbent assay (ELISA). The surface of 96-well plates (flat-bottom Nunc MaxiSorp®) was coated with either transferrin receptor (Abcam) or α-synuclein for 1 h at 37°C. After blocking with bovine serum albumin (BSA), the immunoliposomes were added to each well, followed by a washing step. The secondary antibody conjugated with peroxidase (Goat anti-Mouse IgG [H+L] Cross Adsorbed Secondary Antibody, HRP conjugate, Thermo Scientific-Pierce Antibodies) was then allowed to react for 45 min at RT. To reveal the presence of the antibodies, a citrate solution was used with citric acid (Sigma-Aldrich), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, Sigma-Aldrich) and H2O2 (hydrogen peroxide solution, Sigma-Aldrich). The color intensity was measured by spectrometry using a spectrometer (Synergy 2 Multi-Mode Microplate Reader, BioTek Instruments Inc.). Liposomes without MAb were used as control. The statistical significance was determined by Student's t-test analysis.
7
Immunoblotting Protein Expression Analysis
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Immunoblotting was performed as previously reported [26 (link)]. Briefly, the whole-cell lysates were prepared with RIPA containing 1% PMSF, protease, and phosphatase inhibitor cocktail (Thermo, USA) on ice. The protein concentration was measured with the BCA method (Beyotime, China). After being mixed with 5× loading buffer, the protein extracts were heated at 95 °C for 10 min. Immunoblotting was performed using antibodies against Transferrin receptor (Abcam, USA), HIF-1α (Cell Signaling, USA), and β-actin (Sigma, USA), serving as the internal reference. The membranes were exposed to the Tanon-5200 Chemiluminescent Image System (Tanon, China) for imaging.
8
Proteomic Analysis of Cellular Signaling
9
Protein Expression Analysis by Western Blot
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Cell lysis was performed as described [49 (link)]. Protein concentration was determined by Bradford (Bio-Rad Laboratories, Hercules, CA, USA). Samples were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories). The following antibodies were used: HRAS (Abcam Cat# ab97488, RRID:AB_10680439); VEGF-B (Abcam Cat# ab51867, RRID:AB_2304198); transferrin receptor (Abcam Cat# ab84036, RRID:AB_10673794); Caveolin (BD Biosciences Cat# 610684, RRID:AB_398009); APT-1 (LYPLA1) (Novus Cat# H00010434-M05, RRID:AB_1146125); tubulin (Sigma-Aldrich Cat# T8328, RRID:AB_1844090).
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