Vdr−/− mice, CD45.2 and CD45.1 B6 mice were purchased from the Jackson Laboratory and maintained in the animal facility at the Massachusetts Institute of Technology. All animal studies and procedures were approved by the Massachusetts Institute of Technology's Committee for Animal Care. Antibodies specific for CD11c (N418), CD11b (M1/70), F4/80 (BM8), MHC-II (M5/114.15.2), CD45.2 (104), CD45.1 (A20), CD4 (HK1.4), CD8 (53–6.7), CD19 (1D3) for flow cytometry were from Biolegend. Antibody for Irf4 (3E4) was from eBioscience. Anti-VDR (D2K6W) for immunochemistry staining was from Cell Signaling Technology. Single cell preparation from different organs, staining of cells with fluorophore-conjugated antibodies and analysis of the stained cells using flow cytometry are as described [38 (link)]. Briefly, cells in single cell suspension were incubated with specific antibodies at 4 °C for 20 min, washed twice, and re-suspended in FACS buffer containing DAPI. Cells were run on BD-LSRII, collecting 20,000 to 100,000 live cells per sample. The data were analyzed by FlowJo.
Anti vdr d2k6w
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-VDR (D2K6W) is a rabbit monoclonal antibody that recognizes the Vitamin D Receptor (VDR) protein. It is designed for use in various applications such as Western blotting and immunohistochemistry to detect and analyze the expression of VDR in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
B6 cd45.1 mice, by Jackson ImmunoResearch (1 mentions)
Protease and phosphatase inhibitor mix, by Roche (1 mentions)
β actin ac 15, by Merck Group (1 mentions)
Immun blot pvdf membrane, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Mini protean tgx, by Bio-Rad (1 mentions)
Anti actin, by Merck Group (1 mentions)
Fusion pulse 6, by Vilber (1 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Nebnext chip seq library prep kit, by New England Biolabs (1 mentions)
Murine gm csf, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti vdr d2k6w
1
Characterization of VDR-Deficient Immune Cells
2
Western Blot Analysis of VDR
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Cells were lysed in 50 mmol/l Tris/HCl (pH 7.5), 150 mmol/l NaCl, 1% NP-40, and 1× protease and phosphatase inhibitor mix (both Roche) for 15 min on ice. Lysates were clarified, and total protein concentrations were measured with BCA (Pierce). Proteins (20 μg) were separated on gradient gels (Mini-PROTEAN TGX; Bio-Rad) and transferred on a Immun-Blot PVDF membrane (Bio-Rad). Unspecific binding was blocked using 5% milk powder in TBST (Tris-buffered saline) for 30 min. Anti-VDR (D2K6W, 1:1000; Cell Signaling Technologies, CST) was incubated in 0.5% milk powder in TBST overnight at 4°C. Membranes were washed three times with TBST and incubated with secondary anti-rabbit IgG, horseradish peroxidase (HRP)-linked Antibody (#7074; CST) for 2 hr. To control for equal loading, membranes was probed for β-actin (AC-15; 1:10,000; Sigma-Aldrich) expression for 2 hr at room temeperature (RT). Protein bands were visualized with Luminol reagent (Pierce) using the ChemiDoc XRS+ system (Bio-Rad).
3
Western Blot Analysis of VDR
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Whole cell lysates were prepared using RIPA buffer (Sigma-Aldrich) and quantified with the Qubit Protein Assay Kit (Thermo Fisher Scientific). Samples were separated by 12% SDS-PAGE and transferred to PVDF membranes, blocked with 5% milk (Sucofin) in TBS buffer with 0.1% Tween for 1 h, and incubated with primary antibodies overnight: anti-VDR ((D2K6W) Cell Signaling Technology, Danvers, MA, USA, clone or anti-actin (Sigma Aldrich). Membranes were incubated with secondary antibodies for 1 h at RT and analyzed using the chemiluminescence system Fusion Pulse 6 (Vilber Lourmat).
4
ChIP-seq Analysis of VDR in Alveolar Macrophages
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Freshly isolated AM (1 × 106) were cultured and expanded in complete RPMI supplemented with 25 ng/mL murine GM-CSF (Peprotech) for 2 weeks. Chromatin immunoprecipitation (ChIP) was performed using the SimpleChIP (Cell Signaling Technology) following the manufacture's manual. Briefly, 20 × 106 AM were treated with formaldehyde at a final concentration of 1% to cross-link DNA-protein complexes. Cells were lysed and DNA-protein complexes were sheared by micrococcal nuclease, followed by precipitation with nonspecific rabbit anti-IgG or Anti-VDR (D2K6W) (Cell Signaling Technology. Eluted and purified ChIP DNA was used to prepare the DNA-sequencing libraries with the NEBnext ChIPSeq library Prep kit (NEB) for Next-Generation Sequencing (Illumina). Raw sequencing data was deposited in GEO with accession ID: GSE124725 . ChIP-seq were analyzed with the software package HOMER [58 (link)].
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