Popliteal LNs were isolated from mice at 48 h post-injection immediately after in vivo MRI studies. The removed LNs were fixed in 4% PFA and laid in 30% sucrose solution (Sigma) overnight. Tissues were embedded in Optimal Cutting Temperature (OCT; Sakura Finetek, Torrance, CA, USA) and sectioned at a thickness of 5 μm (Leica). Cryo-sectioned LNs were prepared, and Prussian blue staining, hematoxylin and eosin (H&E) staining and immunofluorescence staining using anti-GFP, anti-myc, and anti-CD25 antibodies (Novus Biologicals, Littleton, CO, USA) were performed. Images were captured and analyzed using the Leica Qwin program.
Anti myc
Manufactured by Novus Biologicals
Sourced in United States, United Kingdom
Anti-myc is a primary antibody used for the detection and analysis of myc-tagged proteins in various research applications. It specifically binds to the myc epitope tag, which is a commonly used protein tag for recombinant protein expression and purification.
Automatically generated - may contain errors
Lab products found in correlation
Sucrose solution, by Merck Group (1 mentions)
Anti gfp, by Novus Biologicals (1 mentions)
Anti pgk1, by Thermo Fisher Scientific (1 mentions)
Immobilon western hrp substrate, by Merck Group (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
Nb600 336ss, by Merck Group (1 mentions)
Anti ha 12ca5, by Cell Signaling Technology (1 mentions)
Anti flag, by Merck Group (1 mentions)
Donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
G box, by Syngene (1 mentions)
Trans blot, by Bio-Rad (1 mentions)
Anti gfp, by BD (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Human ghrelin, by Phoenix Pharmaceuticals (1 mentions)
Rpmi 1640 culture medium, by Thermo Fisher Scientific (1 mentions)
Hepes buffered phenol red free medium, by Thermo Fisher Scientific (1 mentions)
Sre luc, by Promega (1 mentions)
Anti ha, by Novus Biologicals (1 mentions)
Orexin a, by Phoenix Pharmaceuticals (1 mentions)
Cre luc, by Promega (1 mentions)
3 protocols using anti myc
1
Characterization of Popliteal Lymph Nodes
2
Western Blot Protein Detection
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Protein samples were resolved on an appropriate percentage SDS-PAGE gel before being transferred to a PVDF membrane using a semi-dry transfer apparatus (TransBlot SD BioRad) at 25 volts for 30 minutes. After transfer, membranes were blocked for 30 minutes using 10% milk in TBS before being incubated with primary antibody (anti-HA (12CA5) 1:1,000, anti-HA (rabbit: Cell Signaling C29F4) 1:2,500, anti-MYC (9E10) 1:1000, anti-MYC (rabbit: Novus NB600-336SS) 1:1,000, anti-FLAG (Sigma M2) 1:1000, anti-GFP (BD Living Colors 632377) 1:1000, anti-PGK1 (Invitrogen 22C5D8) 1:1000) for one hour at room temperature. Membranes were then washed three times for ten minutes each with 0.1% TBS-Tween20 (TBST) before being incubated with the appropriate secondary antibody in 1% milk in TBST for 30 minutes at room temperature. Incubation with secondary antibody (donkey anti-mouse IgG (Jackson ImmunoResearch) 1:10,000 or donkey anti-rabbit IgG (ImmunoResearch) 1:20,000) was then followed by one brief rinse in TBST and another three washes for ten minutes each with TBST before being incubated with Immobilon Western HRP substrate (Millipore) for 5 minutes and imaged using the G-Box from SynGene. Densitometry was conducted using ImageJ.
3
Mammalian Expression Vector Characterization
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The mammalian expression vector pcDNA3.1(+) was purchased from Invitrogen Life Technologies (Paisley, United Kingdom). The plasmids pcDNA3.1-OX1R, which contain human OX1R cDNA, and pcDNA3.1-GHSR1a, which contains GHSR1a, were obtained from the cDNA Resource Centre (Bloomsburg University, Bloomsburg, PA, United States). pEYFP-N1, containing EYFP, and pRluc-N1, containing the Rluc plasmid, were provided by Clontech and PerkinElmer, Inc. SRE-luc, CRE-luc and NFAT-RE-luc were obtained from Promega (Madison, WI, United States).
Human ghrelin and orexin-A were purchased from Phoenix Pharmaceuticals (Belmont, CA, United States). HEPES-buffered phenol red-free medium, DMEM (Dulbecco’s Modified Eagle Medium) and RPMI-1640 culture medium were purchased from Gibco, (Gibco, Invitrogen, Paisley, United Kingdom). Forskolin (FSK) was purchased from Sigma-Aldrich Shanghai Trading Co. Ltd. (Shanghai, China). Anti-Myc, Anti-HA, Anti-GHSR1a and anti-OX1R antibodies were obtained from Novus Biologicals (Abingdon, United Kingdom). Anti-HA-agarose was purchased from Pierce Chemical Co.
Human ghrelin and orexin-A were purchased from Phoenix Pharmaceuticals (Belmont, CA, United States). HEPES-buffered phenol red-free medium, DMEM (Dulbecco’s Modified Eagle Medium) and RPMI-1640 culture medium were purchased from Gibco, (Gibco, Invitrogen, Paisley, United Kingdom). Forskolin (FSK) was purchased from Sigma-Aldrich Shanghai Trading Co. Ltd. (Shanghai, China). Anti-Myc, Anti-HA, Anti-GHSR1a and anti-OX1R antibodies were obtained from Novus Biologicals (Abingdon, United Kingdom). Anti-HA-agarose was purchased from Pierce Chemical Co.
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