The tumor tissue sections embedded in paraffin after fixing with 10% formalin were cut in 4 µm-thick slices. The paraffin sections were baked at 60 °C for 2 h, followed by deparaffinization. Deparaffinized sections were incubated with 10 mM citrate buffer for antigen retrieval. After endogenous peroxidase had been blocked with 3% hydrogen peroxide at RT for 10 min, the tumor sections were incubated with the following primary antibodies against 4-HNE (bs6313R, Bioss, China), Ki67 (27309-1-AP, Proteintech, USA), CHAC1 (15207-1-AP, Proteintech), or SLC7A11 (DF12509, Affinity, USA) at 4 °C overnight. Next, goat anti-rabbit IgG/HRP as the secondary antibody (PV6001, GoldenBridge, China) was incubated at RT for 20 min. Then, 3,3-diaminobenzidine (DAB) staining was employed and subsequent hematoxylin counterstaining, followed by sealing with neutral gum. The sections were dried at 37 °C overnight and the images were captured under a fluorescent microscope (Olympus).
Df12509
Manufactured by Affinity Biosciences
Sourced in China, United States
DF12509 is a compact bench-top centrifuge designed for general laboratory applications. It features a brushless DC motor and can achieve a maximum speed of 12,000 rpm, with a maximum relative centrifugal force (RCF) of 12,500 x g. The centrifuge has a rotor capacity of 12 x 1.5/2.0 mL microtubes. The unit is equipped with an easy-to-use digital control panel for setting speed, time, and temperature.
Automatically generated - may contain errors
Lab products found in correlation
Df6701, by Affinity Biosciences (4 mentions)
Df6278, by Affinity Biosciences (3 mentions)
Ab125066, by Abcam (3 mentions)
Af7021, by Affinity Biosciences (2 mentions)
Df4255, by Affinity Biosciences (2 mentions)
Ab155282, by Abcam (2 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Bs 6313r, by Bioss Antibodies (1 mentions)
Bca assay kit, by Solarbio (1 mentions)
Anti gapdh, by ABclonal (1 mentions)
β actin ac026, by ABclonal (1 mentions)
A11243, by ABclonal (1 mentions)
A5597, by ABclonal (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ma0186, by Meilun (1 mentions)
Chemidoc camera, by Bio-Rad (1 mentions)
Hrp goat anti rabbit igg, by EarthOx (1 mentions)
Af3293, by Affinity Biosciences (1 mentions)
Lip 1, by Topscience (1 mentions)
12 protocols using df12509
1
Immunohistochemistry of Paraffin-Embedded Tissues
2
Protein Expression Analysis in Cells
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RNA levels were measured by qPCR analysis following to the manufacturer’s instructions. Western blot analysis was also performed according to the manufacturer’s protocol. Briefly, cells or tissues were lysed in RIPA buffer. The protein concentrations were then normalized using a BCA assay kit (Solarbio, Beijing, China). Anti-GAPDH (1:1000, ABclonal, Wuhan, China, AC001), anti-STK33 (1:1000, Abcam, Cambridge, MA, USA, ab206296), anti-MDR1 (1:1000, Affinity, Jiangshu, China, AF5185), anti-GSS (1:1000, Affinity, DF6214), anti-MDM4 (1:1000, Affinity, DF8676), anti-P450 3A4/5 (1:1000, Affinity, AF5312), anti-E-cadherin (1:1000, Affinity, AF0131), anti-SLC7A11 (1:1000, Affinity, DF12509), anti-GPX4 (1:1000, Affinity, DF6701), anti-FTH1 (1:1000, Affinity, DF6278), anti-FSP1 (1:1000, Affinity, DF6516), anti-KRas (1:1000, Affinity, DF6324), and anti-ubiquitin antibodies (1:1000, Affinity, AF0289) were used in this study.
3
Oxidative Stress Marker Analysis
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Antibodies against ALDH2 (A11500), AnxA1 (A1118), ICAM-1 (A5597), GPX4 (A11243), nuclear factor-κB p65 (p65, A16271), β-actin (AC026), and horseradish peroxidase- (HRP-) conjugated anti-rabbit IgG antibody (AS014) were purchased from ABclonal, Inc. (Wuhan, China). Antibodies against phosphorylated p65 (Ser 536, p-p65, bs-0982R) and 8-hydroxy-2′-deoxyguanosine (8-OHdG, bs-1278R) were purchased from Bioss (Beijing, China). Antibodies against SLC7A11 (DF12509), NCOA4 (DF4255), and FTH1 (DF6278) were purchased from Affinity (Liyang, China). Rat tumor necrosis factor-α (TNF-α, E-EL-R2856c) and interleukin-6 (IL-6, E-EL-R0015c) ELISA kits were obtained from Elabscience Biotechnology Co., Ltd. (Wuhan, China). Glutathione (GSH) assay kit (A006-2-1), amylase assay kit (C016-1-1), myeloperoxidase (MPO) assay kit (A044-1-1), malondialdehyde (MDA) assay kit (A003-1-2), and tissue Fe2+ assay kit (A039-2-1) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
4
Western Blot Analysis of Apoptosis Regulators
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We collected U251 cells and tissue samples and used RIPA lysis buffer (89900, Thermo Fisher Scientific, USA) to extract total proteins. Then measured them using a bicinchoninic acid (BCA) Protein Assay Kit (23227, Thermo Fisher Scientific, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis separated the protein samples (n = 3), followed by their transfer onto polyvinylidene fluoride membranes. After 2 h of blocking in 5% non-fat milk, membranes were probed overnight at 4 °C with primary antibodies against FLAG (66008-4-Ig, proteintech, China, 1:5000), BAX (50599-2-Ig, proteintech, China, 1:2000), BCL-2 (68103-1-Ig, proteintech, China, 1:2000), GPX4 (ab125066 abcam, USA, 1:1000); SLC7A11 (DF12509, Affinity, USA, 1:1000), GAPDH (AF7021, Affinity, USA, 1:1000), and 2 h of secondary antibody (PR30011/PR30012, proteintech, China, 1:5000) incubation. Proteins were visualized by enhanced chemiluminescence (MA0186, Meilunbio, China) on a ChemiDoc camera (Bio-Rad), and protein expression levels were quantified using the ImageJ software (v1.8.0). Full and uncropped western blots are presented in Supplemental File.
5
Ferroptosis Regulators and Cellular Signaling
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Bavachin was purchased from MCE (China), and pifithrin-α (PFT-α), deferoxamine (DFO), ferrostatin-1 (Fer-1), and liproxstatin-1 (Lip-1) were purchased from Topscience (China). Vitamin E was purchased from Beyotime (Shanghai, China). Rabbit polyclonal anti-transferrin receptor antibody (TFRC, AF5343, 1 : 1000), rabbit polyclonal anti-divalent metal transporter-1 antibody (DMT1, DF12740. 1 : 1000), rabbit polyclonal anti-ferritin light chain antibody (FTL, DF6604, 1 : 1000), rabbit polyclonal anti-ferritin heavy chain antibody (FTH, DF6278, 1 : 1000), rabbit polyclonal anti-SLC7A11 antibody (DF12509, 1 : 1000), rabbit polyclonal anti-P53 antibody (AF0879, 1 : 1000), rabbit polyclonal anti-STAT3 antibody (AF6294, 1 : 1000), rabbit polyclonal anti-p-STAT3 (705) antibody (AF3293, 1 : 1000), and rabbit polyclonal anti-glutathione peroxidase-4 antibody (GPX4, DF6701, 1 : 1000) were purchased from Affinity Bioscience (China). HRP goat anti-rabbit IgG was purchased from Earthox (USA).
6
Western Blot Analysis of Ferroptosis Regulators
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Western blotting was performed as previously described6 (link). Briefly, cells were lysed using RIPA buffer (Beyotime) with protease and phosphatase inhibitor cocktail (Beyotime), and the proteins were boiled in 5× SDS-PAGE loading buffer (EpiZyme Biotech, China) for 10 min at 100 °C. About 15–20 μg proteins were separated using SDS-PAGE (EpiZyme Biotech) and transferred onto polyvinylidene fluoride membranes (Merck-Millipore, USA). The membranes were blocked with 5% nonfat milk and incubated overnight at 4 °C with antibodies against ARG2 (1:1000, A19233, Abclonal, USA), ODC1 (1:1000, A3898, Abclonal), GPX4 (1:1000, DF6701, Affinity, USA), ACSL4 (1:1000, abs106075, Absin, China), SLC7A11 (1:1000, DF12509, Affinity), GAPDH (1:2000, AF0006, Beyotime), PAOX (1:1000, abs139256, Absin), SMOX (1:1000, abs151305, Absin), catalase (1:1000, A18018, abclonal), c-Myc (1:1000, A1309, Abclonal), TSG101 (1:1000, A1692, Abclonal), CD63 (1:500, abs149061, Absin), GM130 (1:2000, A5344, Abclonal), CD81 (1:1000, A4863, Abclonal). After the membranes were washed with Tris-buffered saline-Tween solution, the secondary antibodies were added to the membranes at room temperature. Finally, the protein bands were visualised with a BeyoECL Plus kit (Beyotime).
7
Immunofluorescence Staining of Lung Tissues
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Lung tissues were paraffin-fixed and incubated overnight at 37°C. Then, these lung sections were deparaffinized and hatched with 3% hydrogen peroxide for 15 min. The slices were heated at microwave treatment and then naturally cooled for 40 min. After performing antigen retrieval, the samples were blocked with 1% BSA for 0.5 h and incubated with primary rabbit anti-NCOA4 (1:100, DF4255, Affinity Biosciences, Jiangsu, China) and anti-SLC7A11 (1:100, DF12509, Affinity Biosciences, Jiangsu, China), respectively, overnight at 4°C. Lastly, homologous fluorescent or biotin-labeled secondary antibodies were incubated with the lung tissues for 2 h at 37°C. The images were captured using a microscope (Nikon, Japan).
8
Western Blot Analysis of Ferroptosis Markers
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After the total protein was extracted, the protein concentration was examined. With 50 μg of total protein, each protein sample was separated for electrophoresis on 8–12% sodium dodecyl sulfate–polyacrylamide gels and transferred onto polyvinylidene difluoride membranes. Then, the membranes would be blocked for 2 h at room temperature and incubated with the primary antibody against FPN1 (bs-4906R, Bioss, China), GPX4 (ab125066, Abcam, UK), ACSL4 (ab155282, Abcam, UK), FTH1 (ab183781, Abcam, UK), TFR1 (ab269514, Abcam, UK), xCT (DF12509, Affinity Bioscience, China), 4-HNE (GTX01087, GeneTex, USA), Nox4 (380,874, Zen-bioscience, China), and β-actin (380,624, Zen-bioscience, China) at 4 °C overnight. Then, the membranes were incubated with the second antibodies (Zen-bioscience, China) for 1 h at room temperature. The target bands were visualized by enhanced chemiluminescence and analyzed on Chemiluminescent gel imaging (MiniChemi 610 Plus, Beijing Saizhi Venture Technology Co., Ltd.), which were then quantified by ImageJ analysis software (National Institutes of Health, Bethesda, MD).
9
Western Blot Analysis of Ferroptosis Markers
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Cells were lysed with radio-immunoprecipitation assay (RIPA) buffer (PC101, Epizyme) containing protease inhibitors to extract total proteins, and bicinchoninic acid (BCA) protein assay kit (ZJ102, Epizyme) was used for protein quantification. Equivalent amounts of proteins (20 μg) from each group were loaded onto 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) gels (G2003-50 T, Servicebio) and then transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were incubated overnight at 4 °C with primary antibodies, including GAPDH antibody (AF7021, Affinity, 1:1000 dilution), SLC3A2 antibody (A19880, Abclonal, 1:800 dilution), SLC7A11 antibody (DF12509, Affinity, 1:1100 dilution), GPX4 antibody (DF6701, Affinity, 1:1000 dilution), ACSL4 antibody (ab155282 Abcam, 1:1500 dilution), and TFR1 antibody (A5865, Abclonal, 1:1000 dilution). After washing with tris buffered saline with Tween-20 (TBST), the membranes were incubated with a secondary antibody Goat Anti-Rabbit IgG (S0001, Affinity, 1:5000 dilution) for 2 h at 37 °C. Signals were detected using a chemiluminescence system (Millipore) and ImageJ software was utilized for the quantification of immunoblotting signals. All experiments were replicated 3 times.
10
Quantitative Analysis of Xenograft Tumor Biomarkers
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A xenograft tumor biopsy was examined using antibodies to Ki-67 (ab15580, 1/100, Abcam) and SLC7A11 (DF12509, 1/100, Affinity) as described in our previous studies [27 (link), 28 (link)]. The staining outcomes were measured utilizing Image-Pro Plus 6.0 (Media Cybernetics, USA) and presented as the proportion of cells exhibiting positivity (for Ki-67) or the average intensity (for SLC7A11). Cell apoptosis in xenograft tumor tissues was detected using the CF488 (Green) Tunel Cell Apoptosis Detection Kit (Servicebio, Wuhan, China) following the guidelines provided by the manufacturer.
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