The haematoxylin and eosin (HE), Picro Sirius Red (PSR) staining and immunohistochemical staining were performed as previously described.19 , 20 Antibodies against CD68 (Abcam), CD45 (Abcam) and 4‐hydroxynonenal (Abcam) were used for immunohistochemical staining.
4 hydroxynonenal
Manufactured by Abcam
Sourced in United States, United Kingdom
4-Hydroxynonenal is a highly reactive aldehyde that is commonly used as a marker for oxidative stress. It is a product of lipid peroxidation and can be detected in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Nitrotyrosine, by Merck Group (2 mentions)
Sorafenib, by Selleck Chemicals (1 mentions)
P bcl 2, by Cell Signaling Technology (1 mentions)
4 phenylbutyric acid 4 pba, by Merck Group (1 mentions)
Ferrostatin 1, by Selleck Chemicals (1 mentions)
Chloroquine cq, by Merck Group (1 mentions)
Ptgs2, by Abcam (1 mentions)
P62 sqstm1, by Proteintech (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
β actin, by Proteintech (1 mentions)
Caspase 3, by Proteintech (1 mentions)
Hepg2, by National Collection of Authenticated Cell Cultures (1 mentions)
Hep3b, by National Collection of Authenticated Cell Cultures (1 mentions)
Plc prf 5, by National Collection of Authenticated Cell Cultures (1 mentions)
Normal goat serum (ngs), by Agilent Technologies (1 mentions)
Hydrogen peroxidase, by Merck Group (1 mentions)
Clone 2a8, by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Timp 1, by Merck Group (1 mentions)
Pecam 1, by Santa Cruz Biotechnology (1 mentions)
12 protocols using 4 hydroxynonenal
1
Histological Staining and Immunohistochemistry
2
Human HCC Cell Line Characterization
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Human HCC cell lines (HepG2, Hep3B and PLC/PRF/5) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cell lines were attached with STR profiling and mycoplasma contamination test reports. HepG2, Hep3B and PLC/PRF/5 have TP53 wild, null and mutant types, respectively. HepG2 cells were maintained in RPMI-1640 medium (HyClone, US), Hep3B and PLC/PRF/5 cells were maintained in MEM medium (HyClone, US). All cell cultures were supplemented with 10% (v/v) fetal bovine serum (FBS) (Bailing, China), 100 U/mL penicillin and 100 μg/mL streptomycin, and incubated in a humidified incubator at 37ºC with 5% CO2.
Chloroquine (CQ) and 4-Phenylbutyric acid (4-PBA) were purchased from Sigma-Aldrich (US). Ferrostatin-1 and Sorafenib were purchased from Selleck (US). Primary antibodies p53, PUMA, NOXA, xCT, PTGS2 and 4-Hydroxynonenal were purchased from Abcam (UK), PERK, ATF4, pBCl-2 and GPX4 were purchased from Cell Signaling Technology (US), β-Actin, Bip, GSK3-beta, DRAM, LC3, SQSTM1 ⁄ p62 and Caspase-3 were purchased from Proteintech (China). Secondary antibodies were all purchased from Proteintech (China).
Chloroquine (CQ) and 4-Phenylbutyric acid (4-PBA) were purchased from Sigma-Aldrich (US). Ferrostatin-1 and Sorafenib were purchased from Selleck (US). Primary antibodies p53, PUMA, NOXA, xCT, PTGS2 and 4-Hydroxynonenal were purchased from Abcam (UK), PERK, ATF4, pBCl-2 and GPX4 were purchased from Cell Signaling Technology (US), β-Actin, Bip, GSK3-beta, DRAM, LC3, SQSTM1 ⁄ p62 and Caspase-3 were purchased from Proteintech (China). Secondary antibodies were all purchased from Proteintech (China).
3
Histological Analysis of Liver Necrosis
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Liver tissue was fixed in 10 % formaldehyde (Sigma-Aldrich), dehydrated, embedded in paraffin blocks, and sectioned into 5 μm slices. The sections were stained with hematoxylin and eosin (H&E; Sigma-Aldrich) for histology, and the necrosis grade was evaluated in 20 random 100 × images per animal, as described by Liu et al. [11 (link)] as follows: “0” indicated normal; “1” indicated necrotic cells in the first cell layer adjacent to the central vein; “2” indicated necrotic cells extending two to three cell layers from the central vein; “3” indicated necrotic cells extending three to six layers from the central vein; “4” indicated necrotic cells extending three to six layers and from one central vein to another; and “5” indicated necrotic cells throughout the section. The sections were deparaffinized and dehydrated for immunohistology, and the endogenous peroxide was inactivated with 3 % hydrogen peroxidase (Sigma-Aldrich). The sections were then blocked with 3 % normal goat serum (DAKO, Glostrup, Denmark) for 1 h, stained with primary antibodies against cytochrome P450 subfamily 2E1 (1:200), 4-hydroxynonenal (1:200, Abcam, Cambridge, MA, USA), or nitrotyrosine (1:50, clone 2A8.2, Millipore, Bedford, MA, USA) for 1 h at 37 °C and then incubated with an horseradish peroxidase (HRP)-detection kit (REALTM EnVision, DAKO) according to the manufacturer’s protocol.
4
Immunohistochemical Analysis of Wound Healing
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The wounds were harvest and fixed as mentioned above and sections were then deparaffinized, rehydrated, and washed in distilled water. The sections were placed in 95~98°C antigen retrieval citrate buffer in a container for 10~15 mins. Endogenous peroxidase activity was blocked by placing the sections in 3% hydrogen peroxide in methanol for 10 mins. Non-specific staining was blocked with normal goat serum, and the sections were incubated with monoclonal mouse anti-rat MMP-2/8/9 and TIMP-1 (Millipore), PECAM-1 (Santa Cruz) CD68 (abcam), MPO (R&D), 4 Hydroxynonenal (abcam) and Anti-pimonidazole MAb1 (Hypoxyprobe, Inc) overnight at 4°C. After washing, a HRP-labeled secondary antibody was applied for 1hr at room temperature then stained with diaminobenzidine, and counterstained with hematoxylin.
5
Immunohistochemical Profiling of Xenograft Tumors
6
Polystyrene Microparticle Assay Protocol
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PS-MPs (1 μm) and fluorescent-labelled PS-MPs (1 μm) were purchasedfrom Tianjin Bestra Chromatography Technology Development Center (25 mg/mL stock solution). The stock solution was diluted to the specified concentration using complete medium. The monoclonal antibodies, p16 (ab51243; 1:50 dilution for IHC, 1:1000 dilution for WB), Ki67 (ab15580; 1:200 dilution), γ-H2AX (ab81299; 1:200 dilution), 53BP1 (ab175933; 1:200 dilution), IL-16 (ab180792; 1:100 dilution), 4-Hydroxynonenal (ab48506; 1:100 dilution), H3K9me3 (ab8898; 1:1000 dilution), Histone (ab1791; 1:1000 dilution), IL-6 (ab9324; 1:1000 dilution), IL-8 (ab18672; 1:1000 dilution), TNF-α (ab1793; 1:1000 dilution), were obtained from Abcam (US). The monoclonal antibodies, α-SMA (19,245; 1:500 dilution), p53 (48,818; 1:100 dilution for IHC, 1:1000 dilution for WB), p21 (2947; 1:50 dilution for IHC, 1:1000 dilution for WB), NF-kB (p65) (8242; 1:500 dilution for IHC, 1:1000 dilution for WB), IL-1β (12,242; 1:500 dilution for IF, 1:1000 dilution for WB), β-actin (4970; 1:1000 dilution), were purchased from Cell Signaling Technology (US). Other reagents were purchased from Thermo Fisher, unless otherwise specified.
7
Protein Extraction and Western Blot Analysis
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Total proteins were extracted in lysis buffer (300 mM NaCl, 50 mM HEPES pH 7.6, 1.5 mM MgCl2, 10 % glycerol, 1 % Triton X-100, 10 mM NaPyrPO4, 20 mM NaF, 1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 1 mM Na4VO3) containing phosphatase inhibitors (all were purchased from Sigma-Aldrich), quantified by protein assay (Bio-Rad, Hercules, CA, USA), separated by 10 % SDS-PAGE (Bio-Rad) and transferred to PVDF membranes (Millipore). After being blocked with 5 % bovine serum albumin (Sigma-Aldrich) in TBS buffer (50 mM Tris–HCl, 150 mM NaCl pH 7.2) with 0.1 % Tween (Sigma-Aldrich) overnight, the membrane was incubated with primary antibody overnight at 4 °C (JNK, phospho-JNK, ERK, phospho-ERK, p38, and phospho-p38 were purchased from Cell Signaling Technology, Danvers, MA, USA 1:2000; cytochrome P450 subfamily 2E1 and 4-hydroxynonenal were purchased from Abcam, 1:1000; and nitrotyrosine and actin were purchased from Millipore at 1:500 and 1:3000, respectively), followed by HRP-conjugated secondary antibody (1:10000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature. The protein intensity was detected with electrochemiluminescence (ECL) reagent (Millipore) according to the manufacturer’s protocol. The western blot band intensity was quantified with the ImageJ software according to the manufacturer’s instructions.
8
Immunofluorescence Imaging of HL-1 Cells
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The HL-1 cells were fixed in 4% formaldehyde for 20 min and were washed with PBS three times. The cells were permeabilized with 0.2% Triton for 10 min. The cells were blocked with 10% goat serum for 1 h following permeabilization. The following primary antibodies were added and incubated overnight: acetylated tubulin (Millipore Sigma); HDAC6 (Cell Signaling, Danvers, MA, USA); 4-Hydroxynonenal (Abcam, Cambridge, MA, USA); LC3 (Cell Signaling); ϒ-tubulin (Abcam); and ubiquitin (Abcam). AlexaFluor 488 goat anti-mouse (ThermoFisher), AlexaFluor 488 goat anti-rabbit (ThermoFisher), or AlexaFluor 594 goat anti-rabbit (ThermoFisher) secondary antibodies were added for 1 h at room temperature. The cell nuclei were stained with DAPI in a VECTASHIELD Mounting Medium (H-1800, Vector Laboratories) and imaged using fluorescence microscopy.
9
Immunohistochemical staining and quantification
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Immunohistochemical staining was performed using the DAKO Envision system (DAKO, Carpinteria, CA, USA), which uses dextran polymers conjugated with horseradish peroxidase to avoid contamination with endogenous biotin. After deparaffinization, tissue sections were treated using a microwave antigen-retrieval procedure in 10 mM sodium citrate buffer. After blocking endogenous peroxidases, sections were incubated with Protein Block Serum-Free (DAKO) to block non-specific staining and were immunostained with antibody against F4/80 or 4-hydroxynonenal (both from Abcam, Cambridge, UK). Stained sections were quantified by iSolution DT 36 software (Carl Zeiss, Oberkochen, Germany), and results were expressed as percentage of the total cells.
10
SERCA2 Immunoprecipitation and Oxidative Modifications
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