Tissue sections from the TMA were deparaffinized, rehydrated through graded alcohols, and washed with phosphate-buffered saline. Antigen retrieval was performed by heating sections in 10 mM citrate (pH 6.0) boiling buffer for 15 min. The tissues were permeabilized with 0.3% Triton X for 1 h at room temperature. After endogenous peroxidase activity was quenched and nonspecific binding was blocked, mouse monoclonal anti-Claudin-2 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-VDR (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-TGR5 antibodies (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated at 4 °C overnight. Biotinylated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was allowed to incubate for 1 h. After washing, sections were incubated with avidin-biotin–peroxidase complex (Vector Laboratories, Burlingame, CA) for 1 h at room temperature. For color reaction development, slides were immersed in Vector NovaRed substrate (Vector Laboratories, Burlingame, CA) for 2 min and counterstained with Flex Hematoxylin for 2 min (Vector Laboratories, Burlingame, CA). A negative control was performed by replacing anti-VDR antibody with normal serum.
Anti vdr
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Italy
Anti-VDR is a laboratory reagent used to detect and analyze the Vitamin D Receptor (VDR) protein. It is a specific antibody that binds to the VDR, allowing for its identification and quantification in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (5 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (3 mentions)
Anti claudin 7, by Thermo Fisher Scientific (3 mentions)
Anti villin, by Santa Cruz Biotechnology (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Anti claudin 5, by Thermo Fisher Scientific (2 mentions)
Immunodetector peroxidase blocker, by Bio SB (2 mentions)
Anti erα, by Bio SB (2 mentions)
Anti puma, by Cell Signaling Technology (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Anti beclin 1, by Santa Cruz Biotechnology (2 mentions)
Anti zo 1, by Thermo Fisher Scientific (2 mentions)
Anti claudin 2, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride (pvdf), by GE Healthcare (2 mentions)
Complete tablet buffer, by Roche (2 mentions)
Biotinylated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Vector novared substrate, by Vector Laboratories (1 mentions)
Anti claudin 2, by Santa Cruz Biotechnology (1 mentions)
Avidin biotin peroxidase complex, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine substrate, by Merck Group (1 mentions)
25 protocols using anti vdr
1
Immunohistochemical Analysis of Claudin-2, VDR, and TGR5
2
Immunohistochemical Analysis of Colonic VDR
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Freshly dissected colon or colon biopsies were fixed overnight with 4% formaldehyde in PBS (pH 7.2), processed, and embedded in paraffin wax. Tissues were cut into 4-μm sections. Colonic morphology was examined by H&E staining. To examine VDR localization in the colon, we stained the slices with anti-VDR (Santa Cruz Biotechnology Inc.) as primary antibodies, followed by staining with horseradish peroxidase-conjugated anti-IgG as second antibodies. Antigen was then visualized with the 3,3′-diaminobenzidine substrate (Sigma-Aldrich) and observed under a light microscope.
3
Colonic Epithelial Cell Protein Analysis
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Mouse colonic epithelial cells were collected by scraping the tissue from the colon of the mouse, including the proximal and distal regions. The cells were sonicated in lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, pH 8.0, 1% Triton X-100) with 0.2 mM sodium ortho-vanadate, and protease inhibitor cocktail. The protein concentration was measured using the BioRad Reagent (BioRad, Hercules, CA, USA). Cultured cells were rinsed twice with ice-cold HBSS, lysed in protein loading buffer (50 mM Tris, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol), and then sonicated. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with primary antibodies. The following antibodies were used: anti-Claudin-5 (Invitrogen, 35-2500, Carlsbad, CA, USA), anti-Claudin-7 (Invitrogen, 34-9100, Carlsbad, CA, USA), anti-VDR (Santa Cruz Biotechnology, SC-13133, Dallas, TX, USA), anti-Villin (Santa Cruz Biotechnology, SC-7672 Dallas, TX, USA), or anti-β-actin (Sigma-Aldrich, A5316, St. Louis, MO, USA) antibodies and were visualized by ECL (Thermo Fisher Scientific, 32106, Waltham, MA, USA). Membranes that were probed with more than one antibody were stripped before re-probing.
4
Immunocytochemical Staining of Cultured Cells
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For immunocytochemical staining, cultured cells were fixed in 4% (vol/vol) paraformaldehyde for 30 min at room temperature, permeabilized in PBST (1x PBS, 0.2% Triton X-100) for 10 min at room temperature, and incubated in sheep serum for blocking. Primary antibodies were incubated overnight at 4 °C, followed by incubation with secondary antibodies overnight at 4 °C. The primary antibodies used in this study included anti-p21 (BD Pharmingen) at a dilution of 1:100, anti-γH2A.X (Abcam) at a dilution of 1:500, and anti-VDR (Santa Cruz Biotechnology) at a dilution of 1:50. Alexa Fluor-coupled secondary antibodies were used (Cell Signaling Technology) at a dilution of 1:500.
5
ChIP Analysis of Immt Promoter
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ChIP analysis was performed according to the manufacturer’s instructions (Millipore, Darmstadt, Germany). Briefly, HepG2 cells were crosslinked with 1% formaldehyde for 10 min at room temperature and then quenched with 125 mM glycine. The cells were resuspended and sonicated in SDS lysis buffer. Lysates were incubated overnight at 4 °C with the following antibodies: anti-mouse IgG (Santa Cruz, Dallas, USA), anti-VDR (Santa Cruz) and anti-RXRα (Santa Cruz). Protein G agarose was added to form immunocomplexes, which were washed and subjected to elution. The samples were treated with RNase A and proteinase K. DNA was subsequently purified using PCR purification spin columns (QIAGEN, Hilden, Germany). The primers used to amplify the Immt promoter regions were designated R1 (−3986 to −3203), R2 (−3157 to −2323), R3 (−2312 to −1724), R4 (−1845 to −1159), R5 (−1179 to −550) and R6 (−574 to 115) from the transcription start site (TSS). The primers are listed in Supplementary Table 1 .
6
Immunohistochemical Detection of Receptor Proteins
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Cultured cells were grown on glass coverslips and fixated in ethanol 96%. Antigen retrieval was done by autoclaving in EDTA (0.1 M, pH 9.0). Slides were blocked with immunodetector peroxidase blocker (Bio SB, Santa Bárbara CA, USA). For EAG1, additional blocking was performed using background Sniper (Biocare Medical, CA, USA). The following primary antibodies were incubated for 2 hours: Anti- ERα (1:250, Bio SB), anti-VDR (1:100, Santa Cruz Biotechnology Inc, CA, USA) and anti-EAG1 (1:300, Novus Biologicals CO, USA). After washing, the slides were sequentially incubated with immuno-Detector Biotin-Link and immuno-Detector HRP label (Bio SB) during 10 minutes each. Staining was completed with diaminobenzidine (DAB) and slides were counterstained with hematoxylin.
7
Western Blot Analysis of Keratinocyte Proteins
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Western blots used protein lysates from keratinocytes seeded in six‐well plates at a density of 500,000 cells/well as described.(76 ) Additional antibodies used for protein detection were anti‐ERp57 (mouse monoclonal; Santa Cruz Biotechnologies), anti‐VDR (mouse monoclonal; Santa Cruz Biotechnologies), anti‐phospho–cyclic AMP response binding element protein (CREB)‐Ser133 (mouse monoclonal; Cell Signaling Technology, Danvers, MA, USA), and anti‐tubulin at 1 μg/mL (mouse monoclonal; Santa Cruz Biotechnologies).
8
Quantitative Western Blot Analysis of Kidney Proteins
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For Western blot analysis, 100 μg of total kidney protein were separated on SDS-polyacrylamide minigels by electrophoresis (21 (link)). After transfer by electroelution to PVDF membranes (GE Healthcare Limited, Little Chalfont, UK), blots were blocked for 1 h with 5% non-fat milk in Tris-buffered saline solution. Blots were then incubated overnight with primary antibodies for anti-VDR and anti-Klotho (1:500 for both; Santa Cruz Biotechnology, Santa Cruz, CA). The labeling was visualized with a horseradish peroxidase-conjugated secondary antibody (anti-rabbit, 1:2,000, or anti-goat, 1:10,000; Sigma Chemical, St. Louis, MO) and enhanced chemiluminescence (ECL) detection (GE Healthcare Limited, Little Chalfont, UK). Kidney protein levels were further analyzed with a gel documentation system (Alliance 4.2; Uvitec, Cambridge, UK) and the software Image J for Windows (Image J-NIH Image). We used densitometry to quantitatively analyze the protein levels, normalizing the bands to β-actin expression (anti-β-actin, Sigma Chemical, St. Louis, MO).
9
Western Blot Analysis of Apoptosis Markers
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HaCat cells and epithelia of biopsies were denatured with Laemmli buffer, centrifuged and undergone a 10-min heat at 95 °C. Proteins in lysates were separated by SDS-PAGE, followed by electrophoretically transferred onto Immobilon-P membranes (Millipore, USA). Analyses of western blot were completed as described25 (link). A series of primary antibodies were utilized, anti-TNFα, anti-VDR and anti-β-actin from Santa Cruz (California, USA); anti-PUMA, anti-caspase 3, anti-caspase 9, anti-Bcl2, anti-Bax, anti-Bak, anti-p53, anti-phospho-NF-κB p65, anti-NF-κB p65 from Cell Signaling (Beverly, MA).
10
Western Blot Analysis of Cellular Signaling
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The cells were lysed on ice using RIPA buffer for 30 minutes by intermittent vortexing and incubation on ice during this period. Protein concentration of whole cell lysates was measured using the Bradford assay (Bio‐Rad). Whole cell lysates (20 μg) for each sample was separated on 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. The separated proteins were transferred onto a polyvinylidene difluoride membrane followed by blocking at room temperature using 5% non‐fat milk diluted in 0.1% TRIS‐buffered saline‐Tween‐20 for an hour. This was followed by an overnight incubation with either anti‐TonEBP (Abcam), anti‐VDR (Santa Cruz), anti‐total p38 (Cell Signaling Technology, Danvers, MA), anti‐phosphorylated p38 (Cell Signaling Technology), anti‐CFTR (Abcam), anti‐tubulin (Cell Signaling Technology), or anti‐beta actin (Santa Cruz) primary antibodies at 4°C. The membranes were washed and incubated with the relevant secondary antibodies conjugated with HRP (anti‐mouse and anti‐rabbit; BosterBio, Pleasanton, CA) for an hour at room temperature. The membranes were washed and incubated with Clarity ECL Western blotting substrate (Bio‐Rad) and resulting chemiluminescence was imaged using Image quant (GE Image Quant LAS 500; GE Healthcare, Chicago, IL). Densitometry analysis was done using ImageJ software (version 6, NIH, Bethesda, MD).
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