Lysates from the cell culture were prepared using RIPA buffer (Wako Pure Chemical Industries). Cells were washed with cold PBS and incubated with RIPA buffer for 15 min on ice. Cell lysates were subsequently transferred into 1.5 ml tubes and centrifuged at 12,000 × g for 20 min at 4°C. Supernatants were transferred to a fresh tube and stored at –80°C before analysis. A total of 10 µg protein was loaded per lane and separated by 10% SDS-PAGE. The expression of pro-IL-1β and β-actin (ACTB) was analyzed by western blot. After transfer onto polyvinylidene fluoride membranes, nonspecific antibody binding was blocked for 1 h at room temperature using Immunoblock (DS Pharma Biomedical, Osaka, Japan). Then, membranes were incubated for 24 h at 4°C with anti-IL-1β antibody (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-ACTB antibody (1:10000, Sigma-Aldrich), followed by an incubation for 1 h with secondary antibody conjugated horseradish peroxidase (HRP; 1:1000, GE Healthcare, UK, Buckinghamshire, UK). Immunoreactive bands were visualized by Western BLoT Quant HRP Substrate (GE Healthcare) using ImageQuant LAS 4000 (GE Healthcare). The results represent at least 3 independent experiments. Quantitative analysis of bands was performed using Image J (National Institutes of Health, Bethesda, MD, USA).
Immunoblock
Manufactured by Sumitomo Pharma
Sourced in Japan
ImmunoBlock is a laboratory equipment designed for use in immunoassay and molecular biology applications. It functions as a platform for conducting various experiments and procedures that involve the manipulation and analysis of biological samples and molecules.
Automatically generated - may contain errors
Lab products found in correlation
Bz 9000, by Keyence (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Anti actb antibody, by Merck Group (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by GE Healthcare (2 mentions)
Western blot quant hrp substrate, by GE Healthcare (2 mentions)
Ripa buffer, by Fujifilm (2 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Anti troponin t, by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Anti il 1β antibody, by Santa Cruz Biotechnology (1 mentions)
Anti lamin a c, by Santa Cruz Biotechnology (1 mentions)
Lsm 5 duo, by Zeiss (1 mentions)
Histovt one, by Nacalai Tesque (1 mentions)
Alexa fluor 594 conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Axiocam hrc, by Zeiss (1 mentions)
Collagen 1, by Bio-Rad (1 mentions)
Mmp 9, by Abcam (1 mentions)
F4 80, by BioLegend (1 mentions)
Cellsens software, by Olympus (1 mentions)
12 protocols using immunoblock
1
Western Blot Analysis of Pro-IL-1β
2
NLRP3 Inflammasome Protein Expression Analysis
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Lysates from the cell culture were prepared using RIPA buffer (FUJIFILM Wako Pure Chemical). The expression of NLRP3, and ACTB were analyzed using SDS-PAGE. After transfer onto
polyvinylidene fluoride membranes, nonspecific antibody binding was blocked for 1 h at room temperature using Immunoblock (DS Pharma Biomedical, Osaka, Japan). Then, membranes were
incubated for 24 h at 4°C with anti-NLRP3 antibody (1:1000, R&D) and anti-ACTB antibody (1:10000, Sigma-Aldrich), followed by an incubation for 1 h with secondary antibody
conjugated horseradish peroxidase (HRP; 1:1000, GE Healthcare, UK, Buckinghamshire, UK). Immunoreactive bands were visualized by Western BLoT Quant HRP Substrate (GE Healthcare)
using ImageQuant LAS 4000 (GE Healthcare). The results represent at least 3 independent experiments. Quantitative analysis of bands was performed using Image J (National Institutes
of Health, Bethesda, MD, USA).
polyvinylidene fluoride membranes, nonspecific antibody binding was blocked for 1 h at room temperature using Immunoblock (DS Pharma Biomedical, Osaka, Japan). Then, membranes were
incubated for 24 h at 4°C with anti-NLRP3 antibody (1:1000, R&D) and anti-ACTB antibody (1:10000, Sigma-Aldrich), followed by an incubation for 1 h with secondary antibody
conjugated horseradish peroxidase (HRP; 1:1000, GE Healthcare, UK, Buckinghamshire, UK). Immunoreactive bands were visualized by Western BLoT Quant HRP Substrate (GE Healthcare)
using ImageQuant LAS 4000 (GE Healthcare). The results represent at least 3 independent experiments. Quantitative analysis of bands was performed using Image J (National Institutes
of Health, Bethesda, MD, USA).
3
Immunofluorescence Analysis of LAMIN A/C and Troponin T in Autopsied Heart Tissue
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Histological sections of the autopsied heart from an 83-year-old patient with EDMD and a 78-year-old control patient were permeabilized with 1% Triton X-100 after deparaffinization, and the antigens were activated with Histo VT one (Nacalai Tesque) at 90 °C for 40 min. After that, cardiomyocyte-containing tissue slices were blocked with ImmunoBlock (DS Pharma Biomedical) at 4 °C overnight and were incubated at 4 °C overnight with each of the following primary antibodies: anti-LAMIN A/C (Santa Cruz Biotechnology) and anti-troponin T (Thermo Fisher Scientific, MA, USA). Preparations were incubated for 1 hour at room temperature with the isotype-specific secondary antibodies: an Alexa Fluor 488-conjugated chicken anti-rabbit IgG antibody and Alexa Fluor 594-conjugated goat anti-mouse IgG antibody, both obtained from Invitrogen. Nuclei were counterstained with 50 ng/mL DAPI (Invitrogen). Fluorescent signals were detected using a fluorescence laser microscope (BZ9000 Keyence, Osaka, Japan) or by confocal microscopy with a ×40/1.2 NA or ×63 × 3/1.2 NA oil-immersion objective (LSM 5 DUO Carl Zeiss, Jena, Germany) just as during the measurement of the cardiomyocyte cytoplasmic and nuclear area.
4
Immunofluorescence Imaging of GL7+ Cells
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Acetone-fixed cryosections (7 μm) were blocked by immunoblock (DS Pharma Biomedical, Osaka, Japan) and then stained with the indicated Abs. The dilution of each antibody is shown in Supplementary Table 1 . Fluorescent images of the sections were captured on a confocal laser scanning system (LSM-780, Carl Zeiss Microscopy GmbH, Göttingen, Germany). GL7+ area in the field was calculated using ImageJ software.
5
Histological Analysis of Liver Tissue
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Liver cryosections (8 μm) were mounted on glass slides, and hematoxylin and eosin and sirius red staining were performed as described.15 Immunostaining was performed by cold acetone fixation. The fixed sections were incubated with ImmunoBlock (DS Pharma Biomedical) and then incubated with Ly6G (BioLegend) or F4/80 (BioLegend) and collagen I (Bio‐Rad, Hercules, CA) or Mmp9 (Abcam) antibodies, followed by Alexa555‐labeled anti‐rat and Alexa488‐labeled anti‐rabbit secondary antibodies (Molecular Probes). Images were captured using Observer Z1 with an AxioCam HRc (Zeiss, Oberkochen, Germany) except for quantification of sirius red‐positive areas, which were analyzed using cellSens software (Olympus, Tokyo, Japan). Ly6G‐positive (+) or F4/80+ cell numbers were counted using Image J software (for Figs. 2 , 3 , 4 , 5 ). Ly6G and Mmp9 double‐positive cell numbers were counted using the BZ‐X700 hybrid cell count software (Keyence, Osaka, Japan).
6
Yeast Cell Labeling with FITC-K4-TAMRA
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After inducing the display, the yeast cells (~ 5 × 106 cells) were collected by centrifugation at 14,000 × g for 1 min at 4 °C, and the pellet was washed with 1 mL phosphate-buffered saline (PBS) containing 5% immunoblock (DS Pharma Biomedical, Osaka, Japan) (PBS-B). After the pellet was resuspended in 100 µL PBS-B, FITC-K4-TAMRA was added at a final concentration of 100 nM, followed by incubation for 15 min at 4 °C, followed by two washings with 1 mL PBS-B.
7
Quantifying Anti-Adenoviral Antibody Levels
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C57BL/6 mice were intravenously administered Ad vectors at 1 × 1010 IFU/mouse. Blood samples were collected via retro-orbital bleeding fourteen days after administration. Anti-Ad antibody levels in the serum were determined by enzyme-linked immunosorbent assay (ELISA). For the ELISA, a 96-well plate was coated with Ad-null (5 × 105 IFU/well) overnight at 4 °C, washed with PBS-0.05% Tween (PBST), and blocked in ImmunoBlock (DS Pharma Biomedical, Osaka, Japan) for 1 hour at room temperature. The serum samples (diluted 1:500) were added to the antigen-coated plate and incubated for 2 hours at 37 °C. The plate was washed with PBST and incubated with biotin-conjugated goat anti-mouse IgG (H+L) (SouthernBiotech, Birmingham, AL) for 2 hours at 37 °C. The plates was then washed with PBST and incubated with streptavidin-HRP (SouthernBiotech) for 1 hour at room temperature. Finally, the plate was washed with PBST and TMB ELISA Peroxidase Substrate (Rockland Immunochemicals, Gilbertsville, PA) was added. The reaction was stopped by the addition of 0.5 mol/l HCl, and absorbance was read at 450 nm on a TriStar LB941 (Berthold Technologies, Bad Wildbad, Germany).
8
Food Allergens and Colitis in Mice
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Serum was collected from 28- to 55-week-old IL-10 KO mice with colitis and 46-week-old BALB/c mice. Serum IgGs against food proteins were examined by enzyme linked immunosorbent assay (ELISA). Food proteins from the NIH-07 standard diet (Oriental Yeast, Tokyo, Japan) (soybean, corn, wheat, fish meal, and skimmed milk) were extracted using phosphate buffered saline (PBS). Microtiter plates were coated with protein extracts and blocked with Immuno Block (DS Pharma Biomedical, Osaka, Japan). Serum samples from IL-10 KO or BALB/c mice were diluted in Immuno Block and then added to the wells in duplicate. Plates were incubated for 2 h at room temperature and then incubated for 1 h at room temperature with antibodies labeled with a horseradish peroxidase (HRP)-conjugated anti-mouse IgG-Fc fragment antibody. After the addition of 3,3′,5,5′-tetramethylbenzidine, the absorbance was measured at 450 nm.
9
Pluripotent Stem Cell Characterization
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Colonies of undifferentiated human iPSCs plated on MEF feeder cells and cardiomyocytes plated on fibronectin-coated dishes were fixed with 4% paraformaldehyde (MUTO Pure Chemicals, Tokyo, Japan) for 30 min at 4 °C. After fixation, cells were permeabilized with 1% Triton X-100 and blocked with ImmunoBlock (DS Pharma Biomedical, Osaka, Japan). Specimens were incubated at 4 °C overnight with each of the following primary antibodies: anti-OCT3/4 (Santa Cruz, CA, USA), anti-NANOG (Abcam, Camb, UK), anti-SSEA3 (Millipore, MA, USA), anti-Tra1-81 (Millipore), anti-TroponinT (Thermo Scientific, MA, USA), anti-alpha-actinin (Sigma-Aldrich), anti-GATA4 (Santa Cruz) and anti-ANP (Santa Cruz). Preparations were incubated with secondary antibodies for 1 h at room temperature. Nuclei were counterstained with 50 ng/ml 4′,6′-diamidino-2-phenylindole (DAPI; Invitrogen). Fluorescent signals were detected using a fluorescence laser microscope equipped with 1.5×105 pixels charged coupled device (CCD) camera (BZ-9000, Keyence, Osaka, Japan).
10
Immunofluorescence Staining of Nuclear Proteins
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The cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (pH 7.0) for 5 min. To stain nuclear proteins, the cells were permeabilized with 0.2% Triton X-100 (Sigma) for 5 min. Subsequently, the cells were treated with ImmunoBlock® (DS Pharma Biomedical, Osaka, Japan) for 30 min at 25℃. The cells were treated with primary antibody in a 1:1 solution of ImmunoBlock® and TBS containing 0.1% Tween-20 overnight at 4℃. Secondary antibody treatment was conducted for 30 min at 25℃. After nuclear staining with DAPI, fluorescence signals were observed via fluorescence microscopy (IX71; Olympus, Tokyo, Japan). Alternatively, fluorescence signals of the cell populations were analyzed via FACS. The primary and secondary antibodies are listed (Table 2 ).
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