Cells were plated in 24-well-plate on coverslips (Electron Microscopy Science, Hatfield, PA) and exposed to nPM/24 h, fixed in 4% PFA/10 min, permeabilized with 1% NP-40/PBS, and blocked with 5% BSA. Primary antibodies were added at 4’C overnight (4-HNE, 1:500, R&D Systems; 3-NT, 1:100, rabbit; Millipore). Immunofluorescence was visualized using Alexa Fluor 488 antibodies (1:400, Invitrogen). Data analysis was blinded for treatment. Images were scored for intensity of expression: individual cells were identified, and intensity of signal normalized on cell size.
4 hne
Manufactured by R&D Systems
Sourced in United States
4-HNE is a reactive aldehyde that is formed during lipid peroxidation. It is a widely used biomarker for oxidative stress and lipid peroxidation in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 antibody, by Thermo Fisher Scientific (1 mentions)
Oxyblot protein oxidation detection kit, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Gpx4 67763 1 ig, by Proteintech (1 mentions)
Nrf2 16396 1 ap, by Proteintech (1 mentions)
Fsx100 microscope, by Olympus (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Apoptag plus peroxidase in situ apoptosis kit, by Merck Group (1 mentions)
8 ohdg, by R&D Systems (1 mentions)
Glutathione (gsh), by R&D Systems (1 mentions)
7 protocols using 4 hne
1
Oxidative Stress Protein Quantification
2
Immunohistochemical Analysis of xCT and 4-HNE
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Paraffin-embedded tissue array or xenografts were cut into consecutive 5 μm sections. The sections were rehydrated and antigen retrieval with microwave was performed. Immunohistochemistry staining was conducted using Streptavidin-HRP Rabbit & Mouse DAB Kit (CwBio, Taizhou, China). In brief, endogenous peroxydase enzyme in the sections was blocked using 0.3% H2O2 for 10 min, followed by PBS washing for 3 times. Thereafter, the sections were subjected to antigen blocking with 5% goat serum for 30 min, followed by incubation with primary antibody at 37 °C overnight. Primary antibodies were used as follows: xCT (Proteintech, Wuhan, China, Cat#26864-1-AP, 1:100), 4-HNE (R&D, Minneapolis, MN, USA, Cat#MAB3249, 1:300). On the next day, the sections were incubated with biotin-labeled secondary antibodies, streptavidin-HRP, and DAB sequentially. Then, sections were subjected to hematoxylin couterstaining for 1 min. The immunostained sections were captured with the 3D HISTECH digital Scanner (Pannoramic MIDI, Budapest, Hungary) at 400 × magnification. The xCT and 4-HNE expression levels were analyzed using the H-score method by two pathologists as previously described [11 (link)]. H-score = 1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+).
3
Oxidative Stress and Autophagy Markers
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Protein was extracted from frozen diaphragm samples, and antibody dilutions were selected according to the manufacturers’ instructions. The following proteins were analyzed as previously described: (1) total and phosphorylated (Tyr705) forms of STAT3 (clones 124H6 and D3A7, Cell Signaling, USA); (2) carbonylated proteins (Oxyblot Protein Oxidation Detection Kit, Millipore, Germany) and 4-hydroxynonenal (4-HNE; R&D Systems, USA) as indices of oxidative stress; and (3) LC3B-I and LC3B-II levels (clone D11, Cell Signaling, USA) as indices of autophagy [8 (link), 18 (link), 19 (link)]. Immunoreactive bands were visualized using enhanced chemiluminescence and Ponceau red for protein loading. Band intensities were quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences, USA), and all values are expressed as n-fold relative to the mean CTL group value.
4
Immunohistochemical Analysis of Oxidative Stress
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Tissue specimens from human or mouse were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned with 6-μm thickness, and immunostained with specific antibodies, including SND1 (10,760–1-AP, Proteintech), Nrf2 (16,396–1-AP, Proteintech), GPX4 (67,763–1-Ig, Proteintech), and 4-HNE (R&D systems, MAB3249). The histological slides were observed under a light microscope (Leica, Germany). The percentage of positive cells was calculated.
TUNEL staining was performed with In Situ Cell Death Detection Kit, POD (Roche, Switzerland) according to the manufacturer’s protocol. Images were acquired with an Olympus FSX100 microscope (Olympus, Tokyo, Japan).
TUNEL staining was performed with In Situ Cell Death Detection Kit, POD (Roche, Switzerland) according to the manufacturer’s protocol. Images were acquired with an Olympus FSX100 microscope (Olympus, Tokyo, Japan).
5
Oxidative Stress Markers in Cortex
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8‐hydroxydeoxyguanosine (8‐OHdG) and 4‐hydroxynonenal (4‐HNE) levels in the cortex of each group (n = 5) were detected by immunohistochemistry. In brief, frozen brain sections were incubated in 0.3% H2O2/methanol. After blocked in a mouse IgG blocking solution, they were incubated at 4ºC with antibodies against 4‐HNE (1:50; R&D Systems) and 8‐OHdG (1:20; R&D Systems), respectively. Immunoreactivity was visualized by a biotinylated secondary antibody, avidin‐biotin‐peroxidase complex, and DAB.
6
Renal Histopathology Assessment Protocol
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Transverse kidney sections were fixed in 10% neutral buffered formalin for 24 h then embedded in paraffin. Tissues sections were deparaffinized with xylene and rehydrated before antigen retrieval using citrate buffer. Tissues were blocked in normal horse serum, followed by incubation with Ki67 (Cat no. 16667, ABCAM) or 4-HNE (Cat no. MA33249, R&D Systems) or NGAL (Cat no. AF1857, R&D Systems). Sections were washed and then incubated with anti-rabbit (or anti-goat or anti-mouse) secondary antibody (Vector Laboratories) following the manufacturer’s instructions. Sections were washed again and exposed to peroxidase substrate, 3,3-diaminobenzidine (DAB) (Vector Labs), washed in distilled water, dehydrated, mounted, and then visualized by light microscopy. Images were analyzed using ImageJ software (National Institutes of Health). The ApopTag Plus Peroxidase In Situ Apoptosis Kit (Cat no. S7101, Sigma-Aldrich) was also used to perform a terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) stain on kidney sections following the manufacturer’s protocol.
7
Oxidative Stress Markers in BALF
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Tracheal intubation was conducted after anesthesia into the rats, followed by three lavages with PBS. Then BALF was collected and centrifuged, and the supernatant was stored at −80°C. The concentrations of GSH, GSSG, 8-OHdG, and 4-HNE (R&D Systems, Minneapolis, MN) were measured in BALF by DAS-ELISA, according to the ELISA kits manufacturer's instructions.
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