Brains and other organs obtained from rats sacrificed on PNDs 5, 10, 15, and 21 rats were frozen immediately in liquid nitrogen and stored at −80 °C. To prepare tissue extracts, organs were minced and sonicated in RIPA lysis buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% sodium deoxycholate, 0.1% Triton X-100, and 0.1% sodium dodecyl sulfate (SDS)), supplemented with protease inhibitor cocktail (Roche, Diagnostics, Mannheim, Germany), containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.2 mM sodium orthovanadate, and 100 mM sodium fluoride. Protein concentrations were determined by BCA protein assay kits (Thermo Scientific, Rockford, IL, USA). Proteins in the extracts were separated by SDS-PAGE and transferred to PVDF membranes (Hybond-LFP, GE Healthcare Life Science, Pittsburgh, PA, USA) using semi-dry blotting system (ATTO, Tokyo, Japan) at 25 V for 15 min as described [13 (link),55 (link)]. After blocking with 1% non-fat milk in PBS containing 0.1% Tween 20 for 1 h, the membranes were incubated overnight at 4 °C with appropriately diluted primary antibody, as above, followed by incubation with appropriate secondary antibodies and visualization with an Odyssey fluorescent imaging system (version 1.2; LI-COR Biotechnology, Lincoln, NE, USA) [55 (link)]. Densitometric analyses on western blot were performed by software Image J as previously described [55 (link)].
Odyssey fluorescent imaging system
Manufactured by LI COR
Sourced in United States
The Odyssey fluorescent imaging system is a state-of-the-art device designed for the detection and quantification of fluorescent signals. It utilizes high-sensitivity optics and advanced imaging software to capture and analyze data from a variety of fluorescent-based assays and applications.
Automatically generated - may contain errors
Lab products found in correlation
Tris glycine sds page gel, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Hybond lfp, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Anti rabbit irdye 800cw, by LI COR (1 mentions)
Donkey anti mouse irdye 680lt, by LI COR (1 mentions)
0.45 μm nitrocellulose membrane, by Bio-Rad (1 mentions)
Rabbit monoclonal anti β actin 13e5, by Cell Signaling Technology (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Bio dot sf apparatus, by Bio-Rad (1 mentions)
Nfat1, by Cell Signaling Technology (1 mentions)
Gapdh nb 600 502, by Novus Biologicals (1 mentions)
Nfat2, by Cell Signaling Technology (1 mentions)
Nf κb p50, by Cell Signaling Technology (1 mentions)
Parp1, by Cell Signaling Technology (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
7 protocols using odyssey fluorescent imaging system
1
Developmental Protein Expression in Rat Organs
2
Immunoblotting Protein Detection Protocol
3
Immunoblotting for SIRT5 protein quantification
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For immunoblotting, cells were lysed in 1X RIPA lysis buffer (ThermoFisher) containing Halt™ Protease and Phosphatase Inhibitor Cocktail (ThermoFisher). Protein concentration was measured using Pierce BCA Protein Assay Kit (ThermoFisher). Cellular lysates were boiled in Laemmli sample buffer for 10 minutes, separated on Tris-glycine/SDS-PAGE gels (Bio-Rad), followed by transfer to 0.45 μm nitrocellulose membranes (Bio-Rad). Membranes were blocked in 5% non-fat milk in TBST buffer for 1 hour at room temperature, then incubated with primary antibodies for 2 hours at room temperature or overnight at 4°C with gentle rocking. Rabbit monoclonal anti-SIRT5 (D5E11, Cell Signaling) and rabbit monoclonal anti-β-actin (13E5, Cell Signaling) antibodies were used at concentrations of 1:1000 and 1:2000, respectively. Membranes were washed three times for 5 minutes before secondary antibody was added for 1 hour at room temperature with gentle rocking. Secondary antibodies used were IRDye®680LT donkey anti-mouse (926–68022, LI-COR) and IRDye®800CW anti-rabbit (926–32213, LI-COR). Membranes were washed three times before being imaged with an Odyssey Fluorescent Imaging System (LI-COR, Lincoln, NE). ImageJ software was used to analyze the optical density quantification of immunoblots.
4
Aspirin Modulates RANKL-Induced Osteoclast Formation
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BMMs were incubated in 6-well plates at a density of 3 × 105 cells/well and treated with RANKL (50 ng/ml) in the presence or absence of aspirin for the indicated time. The cells were rinsed three times with pre-cooled PBS and lysed with RIPA buffer containing 1% protease and phosphatase inhibitors for 30 min on ice. The cell lysate was centrifuged at 12,000 g for 15 min to collect the protein. A bicinchoninic acid assay kit was used to measure protein concentrations. Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Non-specific binding was blocked with 5% BSA for 1 h at room temperature. The membranes were then incubated with primary antibodies at 4 °C overnight. The membranes were then incubated with secondary antibodies for 1 h at room temperature. Images were captured with an Odyssey fluorescent imaging system (LI-COR Biosciences, Lincoln, NE, USA).
5
Quantification of Genomic 5-Methylcytosine
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Genomic DNA of RAW 264.7 was extracted by DNA extraction buffer (50 μl TE buffer, 450 μl STE buffer,10 μl 20% SDS,10 μl protein K (10 mg/ml)). The DNA samples (2 μg per dot) were spotted on a nitrocellulose membrane in a Bio-Dot SF apparatus (Bio-Rad). The membrane was baked at 80 °C for 2 h, blocked in 5% skimmed milk in TBS containing 0.1% Tween 20, and then incubated with 1:1000 dilution of 5-meC antibody (Beyotime, AF5722) overnight at 4 °C, followed by its detection using secondary antibody. Odyssey Fluorescent imaging system (LI-COR Biosciences) was used for visualization. For the staining of total DNA, the blot membrane was hybridized with 0.2% methylene blue (Leagene, DZ0094).
6
Quantitative Immunoblot of Anti-GBS67 Antibodies
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Test sample and controls (2 mg/mL) spotted on nitrocellulose membranes and the membranes were allowed to dry for 20 minutes. Non-specific sites were blocked by soaking the membranes in PBS-5% w/v BSA-0.05% v/v Tween20 10 cm Petri dishes for 1 hour at RT. After 3 washes with PBS 0.05% v/v Tween20, membrane was incubated with either anti-GBS67 serum diluted in 1:1000 with PBS-5% w/v BSA-0.05% v/v Tween20 (positive sera) or serum from non-immunised mice (1:1000 in PBS-5% w/v BSA-0.05% v/v Tween20) (negative sera) for 1 hour at RT. Membranes were washed three times with PBS 0.05% v/v Tween20 (3 x 5 minutes). Finally, membranes were incubated with fluorescent antimice IgG secondary antibody (1:10000 in PBS-5% w/v BSA-0.05% v/v Tween20) for 45 minutes at RT.
After final washing with PBS 0.05% v/v Tween20 (3 x 5 minutes), membranes were scanned using Odyssey fluorescent imaging system (LI-COR Biosciences, Lincoln, NE, USA).
After final washing with PBS 0.05% v/v Tween20 (3 x 5 minutes), membranes were scanned using Odyssey fluorescent imaging system (LI-COR Biosciences, Lincoln, NE, USA).
7
Isolation and Analysis of Nuclear Proteins
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T cells were collected and lysed with nuclear isolation buffer (10 mM Hepes-KOH, pH 8.0, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM dithiothreitol, and 1× protease inhibitors) for 10 min with shaking every 3 min. The nuclear pellet was collected by centrifugation and was suspended and washed with nuclear isolation buffer twice. The nuclear pellet was directly lysed in SDS sample buffer with 8 M urea. SDS-PAGE was performed in the Bolt Bis-Tris system (Thermo Fisher Scientific). Transfer was performed with 120 V for 90 min to a polyvinylidene difluoride membrane. Blocking of the membrane was performed with Odyssey Blocking Buffer (#927-50000; LI-COR) for 60 min at RT with shaking. The antibody incubation was performed at 4°C overnight with an orbital shaker. Antibodies to cFOS (#2250), JUNB (#3753), NFAT1 (#5861), NFAT2 (#8032), NF-κB p50 (#12540), and PARP1 (#9532) from Cell Signaling; NF-κB p65 (c15310256) from Diagenode; and GAPDH (NB600-502) from Novus were used as primary antibodies. Membranes were washed with Tris-buffered saline, pH 7.5, 0.1% Tween for 5 min three times and were incubated with IRDye secondary antibodies in 1/15,000 dilution in blocking buffer for 60 min at RT with shaking. Membranes were washed with TBS-Tween for 5 min three times again and with TBS once. Images were taken with an Odyssey Fluorescent Imaging system (LI-COR).
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