The inferior lobe of the lung tissues was homogenized in RIPA lysis buffer (Thermo Fisher Scientific, Inc.). After that, 30 μg protein was separated in 10% SDS-PAGE. Proteins were then transferred to PVDF membranes (Millipore). Then, the PVDF membranes were blocked with 5% non-fat dry milk for 1 h at room temperature and then incubated with primary antibodies against p-P65 (1 : 1000, AF2006, Affinity); P65 (1 : 1000, AF5006, Affinity); p-IKBα (1 : 1000, AF2002, Affinity); IKBα (1 : 1000, AF5002, Affinity); NLRP3 (1 : 1000, DF7438, Affinity), ASC (1 : 2000, sc-514414, Santa Cruz Biotechnology, INC), Caspase-1 p20 (1 : 2000, AF4005, Affinity), Pro-GSDMD (1 : 1000, AF4012, Affinity), GSDMD p30 (1 : 1000, DF12275, Affinity), IL-18 (1 : 1000, DF6252, Affinity), IL-1β (1 : 1000, AF5103, Affinity), and β-actin (1 : 5000, AF7018, Affinity) at 4°C overnight. Then, the membranes were then incubated with the appropriate secondary antibodies at room temperature for 2 h. β-actin was selected as the loading control. Finally, the protein bands were caught by using a Chemiluminescence image analysis system (Tanon, Shanghai, China) and analyzed with the Image J software (National Institutes of Health, USA).
Df12275
Manufactured by Affinity Biosciences
Sourced in United States
The DF12275 is a laboratory centrifuge designed for general-purpose applications. It is capable of separating samples based on their density differences. The centrifuge features a compact and durable design to accommodate a variety of sample volumes and rotor types.
Automatically generated - may contain errors
Lab products found in correlation
Af4005, by Affinity Biosciences (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Af5002, by Affinity Biosciences (1 mentions)
Af2006, by Affinity Biosciences (1 mentions)
Df6252, by Affinity Biosciences (1 mentions)
Af2002, by Affinity Biosciences (1 mentions)
Af4012, by Affinity Biosciences (1 mentions)
Af5103, by Affinity Biosciences (1 mentions)
Df7438, by Affinity Biosciences (1 mentions)
Af7018, by Affinity Biosciences (1 mentions)
Af5006, by Affinity Biosciences (1 mentions)
Sc 514414, by Santa Cruz Biotechnology (1 mentions)
Semi dry transfer system, by Bio-Rad (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ab16329, by Abcam (1 mentions)
Ripa buffer, by Roche (1 mentions)
Sc 32233, by Santa Cruz Biotechnology (1 mentions)
Optical microscope, by Olympus (1 mentions)
5 protocols using df12275
1
Lung Tissue Protein Analysis
2
Western Blot Analysis of GSDMD, Caspase-1, and GAPDH
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Total protein extracts were prepared by a RIPA Buffer plus protease inhibitors (Roche). Membrane protein extracts were prepared by using a ProteoPrep® Membrane Extraction Kit (p0033, Beyotime, China). Separated the lysates containing equal amounts of protein by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes by a semi-dry transfer system (Bio-Rad). Blots were blocked with 5% normal goat serum in TBST for 2 h at 25°C, then probed with rabbit anti-GSDMD (1.5 µg/mL, DF-12,275, Affinity, USA), rabbit anti-caspase-1/d (1.5 µg/mL, ab16329, Abcam), and rabbit anti-GAPDH (1:800, sc-32,233, Santa Cruz) at 4°C overnight. Then, the blots were visualized on an Odyssey Infrared Imaging System (Li-Cor Biosciences).
3
Immunohistochemical Analysis of GSDMD Expression
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Blocked sections with 0.3% H2O2, followed by preincubation with 5% normal serum and incubation with primary anti-GSDMD (2.5 µg/mL, DF-12,275, Affinity, USA), overnight at 4°C. The sections were incubated with the biotinylated secondary antibody, and then the sections were incubated with streptavidin-horseradish peroxidase and diaminobenzidine, and stained with hematoxylin. The staining intensities of GSDMD were determined by measurement of MOD, using Image-Pro Plus System.
4
Immunohistochemical Analysis of NLRP3 Inflammasome
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Myocardium embedded in paraffin was sliced into 5 µm slices, followed by dehydration in graded alcohol and deparaffinization in dimethylbenzene. After antigen retrieval and blocking, the sections were incubated with primary antibodies against the following targets at 4 °C overnight: NLRP3 (Immunoway, YT5382), ASC (Immunoway, YT0365), cleaved-caspase-1 (Affinity, AF4005), and cleaved N-terminal GSDMD (Affinity, DF12275). The next day, added dropwise with the secondary antibody and added with a streptavidin-peroxidase solution for 10 min. Then, diaminobenzidine (DAB) was used to develop color and hematoxylin was used to reverse stain the nucleus. Finally, the sections were observed with an optical microscope (Olympus, Japan). ImageJ software (NIH, USA) was used to quantify the percentage of positive staining in IHC examinations.
5
Immunofluorescence Assay for GSDMD-N
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Caco‐2 cells were seeded on six‐well plates and exposed to experimental procedures. After washing, the cells were exposed to 4% paraformaldehyde for fixation as well as 0.2% Triton X‐100 for permeation. After that, cells were blocked with 5% bovine serum albumin solution for 1 h, and then cells were incubated with primary antibody against GSDMD‐N (DF12275, Affinity Biosciences) overnight at 4°C, following which was the probe with secondary anti‐rabbit IgG‐Alexa Fluor 488 antibody (#4412, Cell Signaling Technology) for 1 h at 37°C in the dark. Finally, DAPI solution was added to the cells away from light. Immunofluorescence photographs were acquired employing a confocal laser microscope (Leica, TCS SP5).
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