Western blotting experiments were performed according to previous studies [40 (link)]. Briefly, proteins (50 μg) were loaded into a 10% SDS-PAGE gel and electrotransferred to a polyvinylidene difluoride membrane (Millipore, USA). Membranes were incubated overnight at 4°C in primary antibodies recognizing Jagged-1 (1 : 1000, Cell Signaling Technology), Notch-1 (1 : 1000, Cell Signaling Technology), Hes-1 (1 : 1000, Cell Signaling Technology), CD31 (1 : 1000 dilution, Abcam), zonula occludens-1 (ZO-1) (1 : 1000 dilution, Abcam), and vWF (1 : 500, Santa Cruz Biotechnology). Membranes were washed in TBS and incubated in secondary antibodies conjugated to horseradish peroxidase (HRP) (1 : 1000, Cell Signaling Technology) for 2 h at room temperature. GAPDH (1 : 1000 dilution, Abcam) was used as an endogenous loading control. The ImageQuant LAS 4000 mini detection system (GE Healthcare, Buckinghamshire, UK) was used for quantified densitometric analysis, and ImageJ software (National Institutes of Health, Bethesda, MD) was used to analyze results.
Imagequant las 4000 mini detection system
Manufactured by GE Healthcare
Sourced in United Kingdom
The ImageQuant LAS 4000 mini detection system is a compact and versatile imaging device designed for analyzing and quantifying fluorescent and luminescent signals in various life science applications. The system provides high-resolution digital imaging capabilities, allowing users to capture and analyze a wide range of sample types, including gels, blots, and microplates.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase hrp, by Cell Signaling Technology (2 mentions)
Gapdh, by Abcam (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Jagged 1, by Cell Signaling Technology (1 mentions)
Notch1, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Bcl 2, by Abcam (1 mentions)
Stratalinker 2400, by Agilent Technologies (1 mentions)
Ecl kit, by GE Healthcare (1 mentions)
Normal mouse igg, by Santa Cruz Biotechnology (1 mentions)
Block ace, by Sumitomo Pharma (1 mentions)
Hybond n nylon membrane, by GE Healthcare (1 mentions)
Hrp conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Electroblotting apparatus, by Bio-Rad (1 mentions)
Ab179445, by Abcam (1 mentions)
Ab181616, by Abcam (1 mentions)
Ab134161, by Abcam (1 mentions)
Ab134175, by Abcam (1 mentions)
Anti ucp1 antibody, by Abcam (1 mentions)
Protein assay rapid kit, by Fujifilm (1 mentions)
6 protocols using imagequant las 4000 mini detection system
1
Western Blotting Analysis of Angiogenic Markers
2
Western Blot Analysis of Cell Signaling
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Western blotting experiments were performed following standard methods [27 (link)]. In brief, the proteins were loaded onto 10% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes (Millipore, USA). The membranes were incubated with primary antibodies against Bax and Caspase-3 (1 : 1,000, Cell Signaling Technology), as well as Bcl-2, NQO-1 (1 : 1000 dilution, Abcam, UK), and Nrf-2 (1 : 1,000, R&D), overnight at 4°C, followed by sequential incubation with the secondary antibodies conjugated with horseradish peroxidase (HRP) (1 : 1000, Cell Signaling Technology), at room temperature for 2 h. GAPDH (1 : 1000 dilution, Abcam, UK) was used as the internal reference. Quantified densitometric analysis was conducted using an ImageQuant LAS 4000 mini detection system (GE Healthcare, Buckinghamshire, UK), and the results were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).
3
DNA Methylation Detection Protocol
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Genomic DNA samples (500 ng) were spotted onto Hybond N+ nylon membranes (GE Healthcare, Tokyo, Japan). The blotted membrane was dampened with TE and then applied to a UV-crosslink procedure (1200 J/cm2, 30 sec) using Stratalinker 2400 (Agilent Technologies, Santa Clara, CA, USA). The membrane was then blocked with 100% BlockAce (DS Pharma Biomedical, Osaka, Japan) for 30 min at room temperature and incubated with a mouse anti-5-methylcytosine (5-mC) monoclonal antibody (1:1,000 dilution, Catalog No 39649, Active Motif, Carlsbad, CA, USA), with a rabbit anti-5-hydroxymethylcytosine (5-hmC) polyclonal antibody (1:10,000 dilution, Catalog No 39769, Active Motif), or with normal mouse IgG (1 μg/mL, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. Thereafter, the membrane was incubated with HRP-conjugated anti-mouse IgG (1:10,000, The Jackson Laboratory, Bar Harbor, ME, USA) for 1 h at room temperature. The chemiluminescent signals were detected using ECL kits (GE Healthcare, Little Chalfont, UK) and an ImageQuant LAS4000 mini detection system (GE Healthcare).
4
Western Blot Analysis of Cell Signaling
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Stable cells were washed twice with PBS and suspended in a lysis buffer (2% Mercaptoethanol, 20% Glycerol, 4% SDS in 100 mM Tris-HCl buffer, pH 6.8). Equal amount of proteins were loaded and separated by SDS-PAGE, and then transferred onto PVDF membrane (Schleicher&Schuell Co, Keene, NH, USA) using an electro- blotting apparatus (Bio-RAD, Hercules, CA, USA). The membrane was blocked with 5% nonfat milk (R&D, Minneapolis, MN, USA) in TBST solution for 1 hour at room temperature, and incubated overnight at 4℃ with specific antibody to B7H3 (1 : 1000, ab134161; Abcam), cyclinD1 (1 : 1000, ab134175; Abcam), Rb (1 : 1000, ab181616; Abcam), E2F1 (1 : 1000, ab179445; Abcam). After three washes in TBST solution, the membrane was incubated with secondary antibody diluted with TBST solution at room temperature for 2 hours. The signals of detected proteins were visualized on Image Quant LAS 4000 mini detection system (GE, Boston, MA, USA). β-actin were used as a loading control.
5
Tissue Homogenization and Protein Analysis
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BAT and eWAT were homogenized in a lysis buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm NaF, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 500 μM Na3VO4, and protease inhibitors. After 30 min of incubation on ice, cell lysates were centrifuged at 20,000×g for 30 min, and supernatants were collected. The protein concentration was measured using the Protein Assay Rapid Kit (Wako, Osaka, Japan), and the lysate samples were used for immunoblotting. The primary antibodies were as follows: anti-Gapdh antibody (Proteintech, Rosemount, IL, USA) and anti-Ucp1 antibody (Abcam, Cambridge, UK). For the secondary antibodies, Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling, Danvers, MA, USA) was used. An Enhanced Chemiluminescence Western Detection Reagent (Nacalai Tesque) was used for signal development. Resultant signals were captured using an ImageQuant LAS 4000 Mini Detection System (GE Healthcare Life Sciences). The density of each band was analyzed using ImageJ.
6
Spinal Cord Erk1/2 Activation Assay
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Tissue samples were collected from lumbar dorsal horns of the spinal cord at 5 min after injection and stored at -80°C. The tissues were homogenized and sonicated in RIPA (Conway Biotech, China). After centrifugation at 12000 g, the supernatants were collected to determine protein concentration by BCA kit (Solarbio, China). Then, equal amounts of protein were separated on SDS PAGE Bis-Tris 5-10% gels (Solarbil, China) and transferred to polyvinylidene fluoride membranes (GE Healthcare). The blots were blocked with blocking buffer (5% nonfat dry milk in TBST and 0.1% Tween-20) for 1 h at room temperature and then incubated with rabbit-anti p44/42 Erk1/2 mAb (1:10,000), rabbit-anti phospho-p44/42 Erk1/2 mAb (1:10,000) and rabbit-anti glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:10,000) overnight at 4°C, followed by incubation in goat-horseradish peroxidase-linked secondary antibodies (1:2000) for 1 h at room temperature. Immunoblots were developed with enhanced chemiluminescence reagents (Conway Biotech, China), and the intensities of protein bands were quantified by the ImageQuant LAS4000 Mini detection system (GE Healthcare, Sweden).
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