Calnexin, VEGFA, CD31, HIF-1 alpha, (AB0037, AB0063, AB0092, AB0112, Sicgen, Portugal), β-actin, α-tubulin (A5316, T6199, Sigma, USA), PPARγ, AKT, p-AKT(Ser473), VEGFR2, PI3K (#2443, #9272, #4058, #2479, #4249, Cell Signaling, USA), ANG-2, F4/80, Perilipin-A, GLUT4, p-IR(Y1361), GLO-I, C/EBPalpha, PGC1alpha, ANGPTL4, AT1, HIF-2 alpha (Ab8452, Ab74383, Ab3526, Ab65267, Ab60946, Ab96032, Ab40764, Ab191838, Ab2920, Ab9391, Ab8365, Abcam, UK), CD11c, CD206 (bs-2058R, bs-2664R Bioss, USA) TIE-2, IRβ (sc-324, sc-57342, SantaCruz Biotechnology, USA), CEL (KH025, TransGenic Inc, Japan).
Ab65267
Manufactured by Abcam
Sourced in United Kingdom
Ab65267 is a laboratory equipment product. It is designed for use in various scientific applications. The core function of this product is to facilitate specific experimental procedures without making claims about its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Ab40764, by Abcam (1 mentions)
Ab8365, by Abcam (1 mentions)
Ab191838, by Abcam (1 mentions)
Ab9391, by Abcam (1 mentions)
Ab8452, by Abcam (1 mentions)
Ab96032, by Abcam (1 mentions)
Ab2920, by Abcam (1 mentions)
A5316, by Merck Group (1 mentions)
Ab74383, by Abcam (1 mentions)
Ab60946, by Abcam (1 mentions)
Ab3526, by Abcam (1 mentions)
T6199, by Merck Group (1 mentions)
Vegfr2, by Cell Signaling Technology (1 mentions)
Ab18180, by Abcam (1 mentions)
Bx46 microscope, by Olympus (1 mentions)
Ultravision quanto detection system hrp dab, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fsx100 imaging system, by Olympus (1 mentions)
β actin, by Proteintech (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
5 protocols using ab65267
1
Comprehensive Protein Expression Analysis
2
Immunohistochemical Quantification of ABCA1 and GLUT4
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IHC for quantification of ABCA1 and GLUT4 expression was carried out manually using monoclonal antibodies specific to ABCA1 (ab18180, 1:50) and GLUT4 (ab65267, 1:800) (Abcam, Cambridge, UK). Staining was carried out using the UltraVision Quanto Detection System HRP DAB (Thermo Fisher Scientific) according to manufacturer’s instructions, and slides were counterstained using hematoxylin. Negative and positive control slides were included in each set of staining. Negative control slides were stained after omitting the primary antibody incubation step. Immunoreactivity of each tissue section was scored manually using Olympus BX46 microscope (Olympus Corporation, Tokyo, Japan) by a pathologist. For ABCA1 expression, scores of 0, 1 and 2 were assigned based on the staining intensity and for GLUT 4, scores of 0, 1, 2 and 3 were assigned.
3
Immunofluorescence Staining of GLUT4
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Muscle sections were extracted for immunofluorescence staining to evaluate the expression of GLUT4. The free-floating sample sections were incubated with anti-GLUT4 antibody (1:500, ab65267, Abcam) overnight. The sections were then washed 3 times for 5 min in PBS. Secondary antibodies were applied to the sections for 1 h at room temperature. GLUT4 antibody was targeted with Alexa Fluor 594-conjugated secondary antibodies (1:200; Invitrogen). DAPI staining for cell nuclei was added to the secondary antibody solution. All steps were performed using 0.3% Triton X-100 in PBS. After staining, images were taken at randomly selected fields using an OLYMPUS FSX100 imaging system.
4
GLUT4, GSK3, and PGSK3 analysis
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After euthanasia, tissues including liver and muscle were removed and homogenized with RIPA using a homogenizer, and the tissue proteins were isolated. Western blotting was performed with antibodies against glucose transporter 4 (GLUT4) (1:1000, ab65267, Abcam) for muscle tissue. The level of glycogen synthase kinase 3 (GSK3) (1:1000, #5080, Cell Signaling Technology) and phosphorylated glycogen synthase kinase 3 (PGSK3) (1:1000, #9331, Cell Signaling Technology) were detected for liver tissue. β-actin (1:5000, 60,008–1-Ig, Proteintech) was used as a reference protein.
5
GLUT4 Translocation Quantification in Myoblasts
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Quantification of GLUT4 translocation to the cell membrane was determined by flow cytometry in myoblasts as previously described (Koshy et al. 2010) (link). In brief, primary human myoblasts (10 5 cells/condition) from obese subjects (n = 5) were treated with either vaspin (100 ng/mL, 15 min) or insulin (100 nM, 15 min). Post-treatment, cells were washed with PBS and stained for GLUT4 (Anti-GLUT4 antibody ab65267, Abcam. 0.05 µg/10 5 cells, diluted in 2% BSA PBS) on ice for 20 min. A second wash with PBS was then preformed, before addition of the secondary antibody (Alexa Flour 488 goat anti-mouse IgG, Invitrogen, Thermo Fisher Scientific. 5 µg/mL diluted in 2% BSA PBS) for 20 min on ice. Cells were then washed in PBS and fixed with paraformaldehyde (Medium A, Life Technologies) for 20 min at RT. Following a single wash in PBS cells were re-suspended in PBS and the relative surface expression of GLUT4 quantified by measuring the mean fluorescence intensity (MFI) of a minimum 1500 cells/condition using an AccuriC6TM bench top flow cytometer (BD Biosciences).
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