Cinnamaldehyde was obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) or in 100% ethanol (Wako, Osaka, Japan) at a concentration of 5 mM. Anti-NRF2 rabbit polyclonal antibody (H-300) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and horseradish peroxidase- (HRP-) linked anti-rabbit IgG antibody (#7074) was purchased from Cell Signaling Technology (Danvers, MA, USA). For Western blotting analysis, an anti-NRF2 rabbit polyclonal antibody (PA5-27882; Thermo Fisher Scientific), anti-HMOX1 rabbit polyclonal antibody (ab137749; Abcam), anti-NQO1 mouse monoclonal antibody (ab28947; Abcam), and anti-GAPDH (14c10) rabbit monoclonal antibody (#2118; Cell Signaling Technology) were used.
Pa5 27882
Manufactured by Thermo Fisher Scientific
The PA5-27882 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed to perform specific functions in a laboratory setting. However, without access to detailed product information, I cannot provide a concise and unbiased description of its core function while maintaining objectivity. Therefore, the description for this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab28947, by Abcam (2 mentions)
Ab10648, by Abcam (2 mentions)
Ab125066, by Abcam (2 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Vectashield containing dapi, by Vector Laboratories (2 mentions)
Ab137749, by Abcam (1 mentions)
Cinnamaldehyde, by Merck Group (1 mentions)
Horseradish peroxidase hrp linked anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Anti nrf2 rabbit polyclonal antibody h 300, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh rabbit monoclonal antibody 14c10, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Abs16, by Merck Group (1 mentions)
Clone d60c5f10, by Cell Signaling Technology (1 mentions)
Dako autostainer link 48, by Agilent Technologies (1 mentions)
Ab48506, by Abcam (1 mentions)
Dab horseradish peroxidase color development kit, by Beyotime (1 mentions)
Ab227828, by Abcam (1 mentions)
Ab177487, by Abcam (1 mentions)
Pa5 27137, by Thermo Fisher Scientific (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
8 protocols using pa5 27882
1
Cinnamaldehyde Activates the NRF2 Pathway
2
Oxidative Stress Regulation Mechanisms
3
Immunohistochemical Analysis of LKB1, NRF2, and NQO1
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Formalin-fixed, paraffin-embedded (FFPE) tissue sections were prepared as per standard protocol for IHC. Epitope retrieval was performed on the Dako PT link (Dako, Denmark). Immunostaining was completed using the automated staining systems, Dako Autostainer Link48 (Dako, Denmark). The LKB1 antibody (1:100, clone D60C5F10, Cell Signaling Technology), NRF2 antibody (1:50, PA5-27,882, ThermoFisher), and NQO1 antibody (1:1000; ab28947; Abcam) were added for 30 min at room temperature. The slides were counterstained with hematoxylin. LKB1/NRF2/NQO1 expression was evaluated in the background non-neoplastic tissue providing an internal negative control. LKB1/NRF2/NQO1 staining was scored as previously described26 (link),27 (link). The staining intensity was graded as 0 (no staining), 1 + (weak), 2 + (moderate), and 3 + (intense). The percentage of stained tumor cells was recorded and the H-score was calculated using the following formula: 1 × (%cells 1 +) + 2 × (%cells 2 +) + 3 × (%cells 3 +). A final H-score of 0 was assigned as negative, 1–100 as weak, 101–200 as medium, and 201–300 + as strong. All slides were evaluated by a pathologist (P.C.) who was blinded from the patients' outcomes.
4
Immunohistochemical Analysis of NPC Biomarkers
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Paraffin-embedded human NPC, adjacent normal tissues and xenograft tumors were deparaffinized, rehydrated and subjected to antigen retrieval. After blocking with 10% normal goat serum, sections were incubated with anti-4-HNE (1:25, ab48506, Abcam), anti-Nrf2 (1:100, PA5-27882, Invitrogen), anti-FGF5 (1:50, PA5-67553, Invitrogen), or anti-FGFR2 (1:100, ab10648, Abcam) antibody at 4 °C overnight. This is followed by the incubation with HRP-conjugated secondary antibodies (31430 or 31460, Invitrogen). Signals were visualized using DAB Horseradish Peroxidase Color Development Kit (P0202, Beyotime). Images were acquired under a microscope. TMA of NPC and nasal polyp tissues was purchased from Tanda Biotechnology company (Wuhan, China) and stained following the IHC protocol.
5
Proteomic Profiling of Neonatal Rat Hippocampus
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Neonatal rat hippocampal tissues were extracted and total protein was obtained using RIPA buffer containing PMSF. The protein concentration was determined using the BCA assay kit (Beyotime, China) on postnatal day 21. Subsequently, the protein loading buffer was added and the samples were denatured at 95°C for 10 min. After that, the proteins were isolated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to an activated PVDF membrane. Following blocking, the membrane was incubated overnight at 4°C with the appropriate concentration of primary antibody. Subsequently, the fluorescently labeled secondary antibody (IRDye700 and IRDye800, goat anti-rabbit) was incubated for 1 h at 37°C. Color rendering was performed using a chemiluminometer (Licor, America). The primary antibodies used were as follows: anti-NeuN (ab177487, abcam), anti-GPX4 (ab125066, abcam), anti-FTH1 (ybs-5907R, YOYOBIO), anti-PTGS2 (ab226869, abcam), anti-ACSL4 (PA5-27137, Invitrogen), anti-Nrf2 (PA5-27882, Invitrogen), and anti-Keap-1(ab227828, abcam).
6
Protein Expression Analysis by Western Blot
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Protein lysates were prepared using RIPA lysis buffer (Beyotime), and quantified using BCA Protein Assay Kit (Beyotime). Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Blots were then blocked with 5% non-fat milk and incubated with primary antibodies at 4 °C overnight, followed by the incubation with secondary antibody (31430 or 31460, Invitrogen). Signals were detected using ECL substrate (Beyotime). Primary antibodies used in western blot as follows: anti-GPX4 (1:1000, ab125066, Abcam), anti-SLC7A11 (1:1000, ab175186, Abcam), anti-α-SMA (1:2000, ab124964, Abcam), anti-vimentin (1:1000, ab92547, Abcam), anti-FAP (1:2000, PA5-99313, Invitrogen), anti-FGF5 (1:1000, PA5-80630, Invitrogen), anti-Keap1 (1:3000, ab119403, Abcam), anti-Nrf2 (1:2000, PA5-27882, Invitrogen), anti-HO-1 (1:3000, ab68477, Abcam), anti-FGFR1 (1:500, ab76464, Abcam), anti-FGFR2 (1:1000, ab10648, Abcam), anti-FGFR3 (1:1000, ab133644, Abcam), anti-FGFR4 (1:1000, ab178396, Abcam) and anti-β-actin (1:2000, ab8226, Abcam) antibodies. Uncropped western blots were shown in Supplemental materials.
7
Nrf2 Translocation Immunofluorescence Assay
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Immunofluorescence staining performed for Nrf2 translocation study were done using polyclonal Nrf2 antibody (PA5-27882, Invitrogen) and Donkey anti-Rabbit IgG Alexa Fluor 488 (A21206, Invitrogen). HEK293 cells for each condition were fixed in 4% paraformaldehyde for 15 minutes and permeabilized in 0.2% Triton-x solution for 1 hour. After blocking the fixed cells for 1 hour with 0.5% Bovine Serum Albumin (BSA) blocking buffer in TBST, Cells were then incubated with primary antibodies diluted in the blocking buffer overnight at 4°C. The next day, cells were washed 3 times with PBS. They were then incubated in a secondary antibody solution containing secondary antibodies diluted in 0.5% BSA in PBS overnight at 4°C. Counterstaining was performed with Vectashield containing DAPI (Vector Labs).
8
Nrf2 Translocation Immunofluorescence Assay
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Immunofluorescence staining performed for Nrf2 translocation study were done using polyclonal Nrf2 antibody (PA5-27882 , Invitrogen) and Donkey anti-Rabbit IgG Alexa Fluor 488 (A21206, Invitrogen). HEK293 cells for each condition were fixed in 4% paraformaldehyde for 15 minutes and permeabilized in 0.2% Triton-x solution for 1 hour. After blocking the fixed cells for 1 hour with 0.5% Bovine Serum Albumin (BSA) blocking buffer in TBST, Cells were then incubated with primary antibodies diluted in the blocking buffer overnight at 4°C. The next day, cells were washed 3 times with PBS. They were then incubated in a secondary antibody solution containing secondary antibodies diluted in 0.5% BSA in PBS overnight at 4°C. Counterstaining was performed with Vectashield containing DAPI (Vector Labs).
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