GREER laboratories supplied HDM; OVA, LPS, and aluminum hydroxide gel from Sigma; mice spirometer (MAX 1320) from Buxco®, USA; Beijing Zhongshidichuang Science and Technology Development Co. Ltd. supplied animal asthma inducing instrument (YLS-8A); centrifuge (5810 R) from Eppendorf®, Germany; image analyzer (Leica Application Suite V4) from Leica, Germany; and Olympus, Japan (Leica®) supplied optical microscope (DMI3000B); 150-mesh cell sieve (Biosharp, BS-100-XBS, China); bronchial epithelial growth medium (Procell, CM-M007, China); DAPI and cytokeratin specific monoclonal antibody (pan-Cytokeratin, Santa Cruz, sc-8018, USA); leukocyte activation cocktail (550583, BD Biosciences, USA); cell viability marker (Fixable Viability Stain 510 antibody, BD Pharmingen); PE-anti-IL-4 (BD Pharmingen); APC-anti-IL-17A (Biolegend); Lipofectamine 3000 (Invitrogen, USA); bicinchoninic acid (BCA), Beyotime, Shanghai, China; DHT (B8214) and 17β-estradiol (C4348) APExBIO, USA; MBD2 antibody (Abcam, Cambridge, USA); eosinophil antibody (anti-ECP, Biorbyt, Cambridge, UK); neutrophil antibody (anti-Gr-1, Biolegend, San Diego, USA); GATA3 (10417-1-AP) and β actin (60008-1-Ig) Proteintech, USA; RORγt (EPR20006) abcam, USA.
Mbd2 antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
The MBD2 antibody is a tool used in research applications to detect and study the methyl-CpG binding domain protein 2. It is a polyclonal antibody that specifically recognizes the MBD2 protein, which plays a role in the regulation of gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Anti gr1, by BioLegend (4 mentions)
Anti ecp, by Biorbyt (4 mentions)
Aluminum hydroxide gel, by Merck Group (1 mentions)
Anti il 17a apc, by BioLegend (1 mentions)
Bicinchoninic acid (bca), by Beyotime (1 mentions)
Anti il 4 pe, by BD (1 mentions)
Max 1320, by Buxco Electronics (1 mentions)
Pan cytokeratin, by Santa Cruz Biotechnology (1 mentions)
Fixable viability stain 510 antibody, by BD (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Application suite v4, by Leica (1 mentions)
P300 antibody, by Abcam (1 mentions)
Protein a sepharose beads, by Merck Group (1 mentions)
Sp1 antibody, by Abcam (1 mentions)
Dab substrate, by Agilent Technologies (1 mentions)
Igg streptavidin biotin complex kit, by Boster Bio (1 mentions)
Spot image acquisition and processing system, by Nikon (1 mentions)
Hif 1α antibody, by Proteintech (1 mentions)
7 protocols using mbd2 antibody
1
Asthma Mouse Model Reagents
2
Chromatin Immunoprecipitation Assay Protocol
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ChIP assay was conducted with technical support by Genecreate (Wuhan, China). The cells were crosslinked with formaldehyde and then quenched with glycine. Then, the cells were centrifugated, washed, and harvested. The cell pellets were lysed in lysis buffer and sonicated. After centrifugation, the supernatant was diluted in dilution buffer, and part of the samples were taken as the input. Then, the immunoprecipitation was done by incubating overnight with MBD2 antibody (Abcam) or the control rabbit IgG antibody. Then, wash buffer was applied. Then, the samples were eluted and the crosslinking was reversed. Finally, the DNA was purified. The immunoprecipitated chromatin was quantified by RT-qPCR. The procedures of RT-qPCR were conducted as previously described. The primer sequences used for ChIP-qPCR are shown in Table 3
3
Chromatin Immunoprecipitation of Transcription Regulators
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HK-2 and MKN45 cells were cross-linked by incubation in 1% formaldehyde-containing medium for 10 mins at 37°C, and sonicated to soluble chromatin. Sp1 antibody (Abcam, Cambridge, MA, USA), MBD2 antibody (Abcam), MeCP2 antibody (Abcam), MBD3 antibody (Abcam), p300 antibody (Abcam), Di-acetyl H3 antibody (Abcam) and Tetra-acetyl H4 antibody (Abcam) were used to precipitate DNA fragments bound by the corresponding elements. The protein-DNA complex was collected using protein A Sepharose beads (Millipore, Darmstadt, Germany) followed by the processes of elution and reverse crosslink. After protease K treatment (Millipore), samples were extracted with phenol/chloroform and precipitated with ethanol. Recovered DNA was resuspended in TE buffer (Millipore) and amplified by PCR. Primers designed for the gene promoter of Ki-67 were as follows: forward 5′-ATGCGTGAGTGGCTCGCCC-3′, reverse 5′-ATGCGTGAGTGGCTCGCCC-3′ (Ibsbio).
4
Lung Histopathological Analysis
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The lungs were fixed with 10% formalin via the trachea, then removed and stored in 10% formalin. Fixed lung tissues were paraffinized and sectioned (5 μm) for hematoxylin and eosin (H&E) staining and immunohistochemistry [MBD2 antibody (Abcam, Cambridge, United States ), MINK1 antibody (Proteintech, Wuhan, China), eosinophil antibody (anti-ECP, Biorbyt, Cambridge, United Kingdom) and neutrophil antibody (anti-Gr-1, Biolegend, San Diego, United States )]. Select stained sections per group were collected and assessed for MBD2, MINK1, eosinophil, and neutrophil protein expression.
5
Quantifying MBD2 Protein in Lung Tissues
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Human lung and mouse lung tissues were stained with rabbit monoclonal MBD2 antibody (1:500, Abcam, Cambridge, UK) and rabbit polyclonal MBD2 antibody (1:100, Santa Cruz Biotechnology, CA, USA), respectively. These proteins were then detected in lung sections with an IgG Streptavidin Biotin Complex kit (Boster, Wuhan, People’s Republic of China) and developed with DAB substrate according to the manufacturer’s protocol (Dako, Glostrup, Denmark). A Nikon Spot image acquisition and processing system (USA) was used for image assessment. The mean integral OD of MBD2 protein staining in the bronchiolar epithelium was measured as follows.32 (link) We used Image-Pro Plus 4.1 professional image analysis software to open all immunohistochemical images from one sample slide. Thereafter, we activated the Magnetic Lasso tool and dragged selection areas only containing bronchial epithelium, then copy and pasted it into a new image. Then, the area of bronchial epithelium and the integral OD of MBD2-positive epithelium were measured. Finally, the mean integral OD of MBD2 protein staining in the bronchial epithelium was the ratio of the integral OD of MBD2-positive epithelium to area of bronchial epithelium. All of the images are assessed with the same parameters.
6
Immunohistochemical Analysis of Lung Tissue
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Lung tissues were stained for immunohistochemistry (neutrophil-specific antibody (anti-Gr1, Biolegend), eosinophil antibody (anti-ECP, Biorbyt), IL-17A antibody (Proteintech), IL-4 antibody (ABBIOTEC), MBD2 antibody (Abcam), and IRF4 antibody (Proteintech)), and 2~4 H&E stained sections per group were chosen under single-blind conditions to detect and localize NEU, EOS, IL-17A, IL-4, MBD2, and IRF4 protein expression. Using a microscope and computer image processing system (Image-Pro Plus 6.0), two pathologists assessed the NEU, EOS, IL-17A, IL-4, MBD2, and IRF4 scores.
7
Histological Analysis of Lung Tissue
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Lungs were first fixed with 10% formalin via the trachea, and then removed and stored in 10% formalin. Fixed lung tissues were paraffinized and sectioned (5 μm) for hematoxylin and eosin (H&E) staining. Lung tissues were stained for immunohistochemistry (neutrophil-specific antibody [0.2 mg/ml, anti-Gr1, Biolegend, San Diego, CA, USA], eosinophil antibody [1 mg/ml, anti-ECP, Biorbyt, Cambridge, United Kingdom], MBD2 antibody [Abcam, Cambridge, United Kingdom] and HIF-1α antibody [Proteintech]). Select stained sections from each group were collected and assessed for neutrophil, eosinophil, MBD2, and HIF-1α protein expression.
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