After 24 h of treatment with different concentrations of the indicated agents, cells were harvested by trypsinization and washed twice with PBS. Next, the cells were incubated for 20 min at 4 °C with mouse anti-human CD261 azide free (DR4) primary antibody (Diaclone, Besancon, France) or normal mouse IgG control (Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:25 in PBS. After being washed twice with PBS containing 1% FBS, cells in 50 μL PBS containing 1% FBS were incubated with 2 μL FITC-goat anti-mouse IgG secondary antibody (Bioss, Beijing, China) for 30 min at room temperature. Then the cells were washed twice and resuspended in 300 μL PBS containing 1% FBS. Flow cytometry was used for detection. Analysis was performed with FACScan software.
Control normal mouse igg
Manufactured by Santa Cruz Biotechnology
Sourced in France, United Kingdom, Switzerland
Control normal mouse IgG is a laboratory reagent used as a control in immunological experiments. It is a purified immunoglobulin G (IgG) fraction derived from the serum of normal, non-immunized mice. This product serves as a negative control to help establish baseline levels and ensure specificity of experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Protein g dynabead, by Thermo Fisher Scientific (2 mentions)
Accuri c6, by BD (1 mentions)
Bioruptor sonicator, by Diagenode (1 mentions)
Chip assay kit, by Beyotime (1 mentions)
N histofine simple stain max po multi, by Nichirei Biosciences (1 mentions)
Heat processor solution ph6, by Nichirei Biosciences (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Roche (1 mentions)
D eclipse confocal laser scanning microscope, by Nikon (1 mentions)
Kgm gold, by Lonza (1 mentions)
Ultracruz mounting medium, by Santa Cruz Biotechnology (1 mentions)
1000 μl tips, by Greiner (1 mentions)
Phenylmethanesulfonyl fluoride, by Merck Group (1 mentions)
9 protocols using control normal mouse igg
1
Flow Cytometric Analysis of DR4 Expression
2
Quantifying Cell Surface Receptor Expression
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An amount of 2.5×105 cells were incubated with primary antibodies in triplicate: mouse anti-CAR clone RcmB (1:500; Millipore, Watford, UK), mouse anti-EGFR clone H11 (1:200; Thermo Scientific), mouse anti-FGFR1 clone M19B2 (1:100; Abcam, Cambridge, UK), and normal mouse IgG control (1:200; Santa Cruz Biotechnology, Heidelberg, Germany) on ice for 1 h, followed by incubation with a secondary goat antimouse IgG Alexa Fluor647-conjugated antibody (1:500; Life Technologies, Paisley, UK) for 30 min on ice. The cells were fixed in ice-cold 2% paraformaldehyde (PFA), 2×104 gated events were acquired on BD Accuri C6 (BD Biosciences) flow cytometer, and data analysis was performed using FlowJo software (Tree Star, Ashland, OR).
3
Quantifying SLINKY Transcript Binding by HNRNPK
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RNA immunoprecipitations (RIP) were done following an Abcam protocol [38 ]. Briefly, ccRCC cells were collected, and nuclei isolated and then lysed in RIP buffer. Chromatin was sheared using a Bioruptor sonicator (Diagenode) set for four 30s cycles. HNRNPK was immunoprecipitated with a mouse anti-HNRNPK antibody (Abcam; 3C2), in comparison to normal mouse IgG control (Santa Cruz Biotechnology). Co-immunoprecipitated SLINKY transcript was then detected and quantified by Q-RT-PCR, as detailed above.
4
ChIP Assay for Transcription Factor Binding
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ChIP assays were performed according to the ChIP Assay Kit (Beyotime) manufacturer instructions. Briefly, ASVC were fixed by adding formaldehyde to a final concentration of 1 % and incubated at 37 °C for 10 min to allow cross-linking of endogenous proteins and DNA. Following three times of wash with cold PBS supplemented with 1 mM PMSF, the cells were resuspended using a buffer containing 1 % SDS and 1 mM PMSF, and lysed by sonication using a sonicator (Scientz-IID, China). After centrifugation, the supernatant was collected and the chromatin in the supernatant was immunoprecipitated with antibodies anti-KLF13 (sc-9605, Santa Cruz) or normal mouse IgG control (Santa Cruz). Input control and the DNA obtained from the immunoprecipitation were amplified by PCR using primers specific to the porcine PPARγ promoter containing KLF13 binding site using the following primers: sense 5′-AACAGGACCTCATTGCTTATC-3′, antisense, 5′-CCACAGCAGGAAC TCACAAT-3′.
5
Quantifying IL-13Rα2 Expression in Atopic Dermatitis Skin
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Eleven lichenified lesional AD skin and 11 normal skin samples were embedded in paraffin by the conventional method and cut into 3-μm-thick sections. Antigen retrieval was performed using Heat Processor Solution pH 6 (Nichirei Biosciences, Tokyo, Japan) at 100 °C for 40 min, and endogenous peroxidase was blocked by incubating the sections with 3% H2O2 (Nichirei Biosciences). The sections were then incubated with anti-IL-13Rα2 (Abcam, 750×) antibody or control normal mouse IgG (Santa Cruz Biotechnology) for 30 min, followed by incubation with the secondary antibody, N-Histofine Simple Stain MAX-PO MULTI (Nichirei Biosciences). Immunodetection was conducted with 3,3-diaminobenzidine as the chromogen, followed by light counterstaining with hematoxylin. The number of IL-13Rα2-positive keratinocytes was counted in three high-power view areas, and the average percent positivity was calculated in each slide.
6
Twist1-DNA Interaction Analysis by ChIP
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CAF cells were grown to 70–80% confluency and fixed with 1% formaldehyde. Nuclear extracts were sonicated to shear the DNA to a length under about 500 bp. Twist1-DNA complexes were immunoprecipitated using Protein G Dynabeads (Invitrogen) conjugated with anti-Twist1 IgG (Abcam, ab50887) or control normal mouse IgG (sc2025, Santa Cruz). After the precipitated DNA was eluted, crosslinks were reversed by 0.3 M NaCl and purified by QIAquick PCR Purification Kit (Qiagen, CA, USA). Then PCR was performed on the purified DNA using primer sets are described in Supplementary Table 2 .
7
Co-Immunoprecipitation of E1B-55k Protein
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Total HEK293 cell lysates were prepared by lysing cells in ice-cold lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 0.5% Nonidet P-40), and complete protease inhibitor cocktail (Merck) for 20 min on ice. For Co-IP, Dynabeads protein G (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. In brief, antibodies (control normal mouse IgG (Santa Cruz Biotechnology; 2 ug) and (E1B-55k (2A6) (Generous gift from P.E. Branton [17 (link)]; 10 μL)) were bound to 50 μL washed beads by incubation for 1 h at room temperature. The antibody-coated beads were then incubated with cell lysate (400 μg total protein) at 4 °C overnight. The beads with immune complexes were washed four times with lysis buffer and two times with PBS/0.02% Tween-20 (Merck) and resuspended in PBS before analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
8
Immunofluorescent Analysis of IL13Rα2 Expression
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Immunofluorescent analysis was performed on cell sheets cultured in 4-well slide chambers (Lab-Tek, Rochester, NY, USA) with KGM-Gold (Lonza) for 48 h, scratched using 1000-μL tips (Greiner Bio-One), and incubated for 6 h at 37 °C in 5% CO2. The cells were washed with phosphate-buffered saline 3 times for 5 min each and fixed in cold acetone for 10 min at room temperature. The cell sheets were blocked with 10% bovine serum albumin (Roche Diagnostics, Basel, Switzerland) and incubated with mouse monoclonal anti-IL13Rα2 (Abcam) or control normal mouse IgG (Santa Cruz Biotechnology). Goat anti-mouse IgG conjugated with Alexa Fluor 488 dye (Thermo Fisher Scientific) was used for the secondary antibody. The nucleus was stained with 4′,6-diamino-2-phenylindole (DAPI). Slides were then mounted with Ultra Cruz mounting medium (Santa Cruz Biotechnology) and were observed using a D-Eclipse confocal laser scanning microscope (Nikon, Tokyo, Japan). The immunofluorescence intensity was measured using ImageJ software.
9
Quantification of RISC-loaded siRNA in Liver
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Measurement of RISC-loaded siRNA (antisense strand) in liver was performed as previously reported by Nair et al. [21] using a combination of immunoprecipitation followed by the stem-loop RT-qPCR procedure described earlier.
Processed (powdered) liver prepared from 50 mg of tissue was resuspended at 100 mg/mL in prechilled lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% Triton-X 100; chemicals were obtained from ThermoFisher Scientific), supplemented with one tablet of cOmpleteÔ Mini, EDTA-free Protease Inhibitor Cocktail (Roche, Sigma-Aldrich) and 1 mM of phenylmethanesulfonyl fluoride (Sigma-Aldrich). Precleared samples (using QAE-Sephadex Ò A-50 resin; Sigma-Aldrich) were then subjected to Ago2 immunoprecipitation by incubation with anti-mouse Ago2 (Clone No: 2D4; FUJIFILM Wako Pure Chemical Corporation, San Diego, CA) and control normal mouse IgG (sc-2025; Santa Cruz Biotechnology, Inc., Dallas, TX) followed by precipitation with Protein G Dynabeads (Life Technologies, ThermoFisher Scientific). Final lysates of RISC-loaded siRNAs were subsequently quantified by the stem-loop RT-qPCR approach described earlier.
Processed (powdered) liver prepared from 50 mg of tissue was resuspended at 100 mg/mL in prechilled lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% Triton-X 100; chemicals were obtained from ThermoFisher Scientific), supplemented with one tablet of cOmpleteÔ Mini, EDTA-free Protease Inhibitor Cocktail (Roche, Sigma-Aldrich) and 1 mM of phenylmethanesulfonyl fluoride (Sigma-Aldrich). Precleared samples (using QAE-Sephadex Ò A-50 resin; Sigma-Aldrich) were then subjected to Ago2 immunoprecipitation by incubation with anti-mouse Ago2 (Clone No: 2D4; FUJIFILM Wako Pure Chemical Corporation, San Diego, CA) and control normal mouse IgG (sc-2025; Santa Cruz Biotechnology, Inc., Dallas, TX) followed by precipitation with Protein G Dynabeads (Life Technologies, ThermoFisher Scientific). Final lysates of RISC-loaded siRNAs were subsequently quantified by the stem-loop RT-qPCR approach described earlier.
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