The frozen sections were incubated with 3% (v/v) H2O2 for 15 min to quench the endogenous peroxidase activity and blocked with 10% goat serum (ZLI-9021, ORIGINE, Beijing, China) for 1 h at room temperature. The sections were incubated with anti-PPARα (1:100 dilution, PA1-822A, Invitrogen, Waltham, MA, USA) antibodies overnight at 4 °C. The biotinylated secondary antibodies of the enzyme-labeled goat anti-mouse/rabbit IgG polymer (GK600505-B, Gene Tech, Shanghai, China) were added, followed by an incubation at 37 °C for 1 h. The slides were then developed with a DAB chromogenic solution (GK600505, Gene Tech, Shanghai, China) and counterstained with hematoxylin. The images were acquired using an Olympus laser scanning microscope (U-LH100-3), and the intensity of the immunostaining was analyzed by the average of 5 slides with 5 random fields at ×40 per slide using Image J software (NIH, Bethesda, MD, USA) [26 (link)].
Pa1 822a
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PA1-822A is a laboratory equipment product. It is designed to perform a specific core function within a laboratory setting. A detailed description of the product's features and capabilities can be provided, while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Gk600505, by Gene Tech (1 mentions)
Ab2961, by Abcam (1 mentions)
α sma, by Abcam (1 mentions)
U lh100 3, by Olympus (1 mentions)
Ab41525, by Abcam (1 mentions)
Sc 9104, by Santa Cruz Biotechnology (1 mentions)
T6557, by Merck Group (1 mentions)
Lf2000, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Pierce protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti pparα, by Thermo Fisher Scientific (1 mentions)
Ab8245, by Abcam (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Anti gapdh, by Abcam (1 mentions)
4 protocols using pa1 822a
1
Immunohistochemical Analysis of PPARα
2
Immunofluorescence Staining of PPARα and αSMA
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The frozen sections were permeabilized with 0.1% Triton X-100 (9002-93-1, Sigma, MA, UK) and blocked with 10% goat serum (ZLI-9021, ORIGINE, Beijing, China) for 30 min at room temperature. The sections were incubated with the following diluted primary antibodies overnight at 4 °C: PPARα (1:100 dilution, PA1-822A, Invitrogen, Waltham, MA, USA) and αSMA (1:200 dilution, ab2961, Abcam, Madison, WI, USA). The fluorescent secondary antibodies, goat anti-rabbit TRITC (ZF-0316, ORIGINE, Beijing, China) and goat anti-mouse FITC (ZF-0312, ORIGINE, Beijing, China), were mixed and added, and the slides were further incubated at 37 °C for 1 h. The sections were then incubated with 4′,6′-diamidino-2-phenylindole (DAPI) and mounted. The images were acquired using an Olympus laser scanning microscope (U-LH100-3, Olympus Corporation, TKY, JPN).
3
Inducible PPARα Transcription Factor Expression
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For transient transfection, 293 T cells were co-transfected with an rtTA plasmid and the Lenti.TRE-PPARαΔ13_PGK-Cre construct using LF2000 (ThermoFisher Scientific), and were treated (or not) with 2 μg ml−1 doxycycline one day later for 24 hr to monitor PPARαΔ13 induction. For all experiments, cells were lysed in RIPA (150 mM NaCl, 20 mM Tris pH 7.4, 0.1% SDS, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, one protease inhibitor cocktail tablet (11836145001, Roche)). Proteins were quantified using a BCA assay, and 50 μg (or 5 μg after Seahorse) was used for western blot. Antibodies used are anti-PPARα (PA1-822A, ThermoFisher Scientific, 1:1000; RRID:AB_2165595 ), anti-GLUT1 (07–1401, Millipore, 1:1000; RRID:AB_1587074 ), anti-GLUT3 (ab41525, Abcam, 1:1000; RRID:AB_732609 ), anti-β-tubulin (sc-9104, Santa Cruz Biotechnology, 1:1000; RRID:AB_2241191 ), and anti-γ-tubulin (T6557, Sigma-Aldrich, 1:10’000; RRID:AB_477584 ).
4
Dentate Gyrus Protein Analysis
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The dentate gyrus was dissected out on ice under dissecting microscope immediately after the behavioral test and homogenized with RIPA lysis buffer (Thermo Fisher Scientific) containing phosphatase inhibitors (Merck Chemicals (Shanghai), Shanghai, China). The Pierce Protein Assay Kit (Thermo Fisher Scientific) was used to determine the protein concentration and a total amount of 30 µg of protein was loaded to the SDS-PAGE gel. The proteins were then transferred to a PVDF membrane followed by incubation in the blocking buffer (5% dry milk and 0.1% Tween 20 in 1× Tris-buffered saline) and subsequently primary antibodies at 4 °C. Primary antibodies used in the study were as follows: anti-PPARα (1:1000, PA1-822A, Thermo Fisher Scientific), anti-Cas9 (1:1000, 19526, CST, Danvers, MA, USA), anti-GAPDH (1:1000, ab8245, Abcam, Cambridge, MA, USA). The membrane was washed and incubated in HRP conjugated secondary antibody the next day. Signals were visualized with the Chemi-Doc XRS+(Bio-Rad; Hercules, CA, USA) and quantified using Image J.
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