Freshly dissected bones were fixed in 4% paraformaldehyde overnight, followed by decalcification in 10% EDTA for 1 week, and then dehydrated using a series of graded ethanol and embedded in paraffin. Samples were then cut into 5-µm-thick longitudinally oriented sections, deparaffinized in xylene, and rehydrated in decreasing concentrations of ethanol followed by distilled water. After deparaffinization and antigen retrieval, sections were blocked in PBS with 5% bovine serum albumin (BSA) for 1 hour and then stained overnight with the following primary antibodies: goat-anti-LepR (R&D: AF497, 10 µg/mL) and rabbit-anti-CD56 (Proteintech: 14255-1-AP, 1:2000). Next, samples were incubated with appropriate secondary antibodies, including donkey anti-goat Alexa Fluor 555 and donkey anti-rabbit Alexa Fluor 647 (all from Invitrogen, 1:400). Slides were mounted with anti-fade prolong gold (Invitrogen) and images were acquired with a Zeiss LSM780 confocal microscope.
Af497
Manufactured by R&D Systems
Sourced in United States
AF497 is a recombinant human Activin A/Inhibin βA Protein. It is a member of the transforming growth factor-beta (TGF-β) superfamily and plays a role in cell growth, differentiation, and apoptosis. The specific function and intended use of this product is not included in this description.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Anti fade prolong gold, by Thermo Fisher Scientific (1 mentions)
Ab19028, by Abcam (1 mentions)
Ecl solution, by Thermo Fisher Scientific (1 mentions)
Streptavidin brilliant violet 421, by BioLegend (1 mentions)
Macs cell separation system, by Miltenyi Biotec (1 mentions)
Nl002, by R&D Systems (1 mentions)
Ebioscience flow cytometry staining buffer, by Thermo Fisher Scientific (1 mentions)
4 protocols using af497
1
Immunohistochemical Analysis of Leptin Receptor and CD56 in Bone Samples
2
Western Blotting Analysis of Leptin-R, Kisspeptin, and GPR54
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Western blotting was performed as described in a prior study.17 (link) Hypothalami and testicles were washed twice in ice-cold phosphate-buffered saline (PBS), and then dissociated in radio-immunoprecipitation assay (RIPA) buffer. The samples were then centrifuged. 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 50 μg of total protein from each sample, which was then transferred to membranes. After blocking, membranes were incubated with anti-Leptin-R antibody (AF497, diluted 1:1000; R&D Systems, Minneapolis, MN, USA), anti-kisspeptin antibody (ab19028, diluted 1:100; Abcam, Cambridge, UK), and anti-GPR54 antibody (abs136643, diluted 1:100; Absin, Shanghai, China). Membranes were then washed and incubated with secondary antibodies (diluted 1:5000; ZSGB-BIO, Beijing, China). Detection was performed by chemiluminescence using ECL solution (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was tested at least in triplicate.
3
Assessing Lipid Uptake in Mesenchymal Stem Cells
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Mice were injected intravenously with 1, 1′-dioctadecyl-3,3,3′3′-tetrametryl-indocarbocyane perchlorate –labeled nLDL (DiI-nLDL) or oxLDL (DiI-oxLDL) 20 μg per mouse, 4 h before euthanasia. For flow cytometry analysis, bone marrow cells were flushed and red blood cells were removed as described above. CD45+ cells were removed by CD45 MicroBeads using MACS cell separation system (Miltenyi Biotec). Flow cytometric analyses were performed using a LSR II flow cytometer. The antibody used was anti-LepR-biotin (R&D Systems, AF497, 1:500) followed by a streptavidin-brilliant violet 421(Biolegend, 405225, 1:500). The uptake of DiI-nLDL and DiI-oxLDL by MSCs were analyzed by calculating the frequency of PE-DiI+ cells in the total CD45-LepR+ cell population.
4
Quantifying Surface Expression of Lepr and Epor
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Surface expression of Lepr was assayed by staining cells using a mouse leptin receptor antibody (R and D Systems, AF497) followed by a NorthernLights NL637-conjugated secondary antibody (R and D Systems, NL002). Surface expression of Epor was assayed using an Epo-Fc fusion (Abcam) labeled with NHS-Cy5 (Sigma). 1 × 106 Ba/F3 cells expressing Epor or Lepr variants were washed 3x and resuspended in eBioscience Flow Cytometry Staining Buffer (ThermoFisher). Prior to all staining steps, the cells were blocked with anti-mouse BD Fc Block (BD Bioscience) for 30 min on ice. Cells were then incubated with murine Lepr antibody or Cy5-Epo-Fc fusion (2.5 μg/106 cells) for 30 min on ice. For Lepr expressing cells, this was followed by another 3x wash in flow cytometry staining buffer, reblocking with anti-mouse BD Fc Block, and a 30 min incubation with NL637-conjugated secondary antibody. All samples were subjected to a final 3x wash and resuspension in 300 uL flow cytometry staining buffer. Surface staining was analyzed using an Accuri C6 flow cytometer.
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