Cells were cultured on coverslips under identical conditions and treatments as described above. The cells were fixed with 4% (wt/vol) paraformaldehyde for 30 min at room temperature, and permeabilized with cold methanol for 20 min. After washed with wash buffer (Beyotime, Nanjing, China) for 3 times, cells were blocked for 1 h at room temperature, and then incubated with primary antibodies anti-Dystrophin (1:500, Abcam, Cambridge, UK) at 4 °C overnight. Secondary antibody with Alexa Fluor 555 (1:500, Beyotime, Nanjing, China) was incubated for 3 h at 4 °C after washed for 3 times. Cell nucleuses were stained with DAPI solution (Beyotime, Nanjing, China). Images were taken using a confocal laser scanning microscope TCS SPE (Leica, Weztlar, Germany). Cells were also observed in bright field at a low magnification (10×, 20×) with a light microscope (NIKON, Tokyo Metropolis, Japan), and measured the diameter of muscle fiber with the software NIS-elements D.
Wash buffer
Manufactured by Beyotime
Sourced in China
Wash buffer is a solution used to rinse and clean laboratory equipment and samples during various experimental procedures. It helps remove contaminants, excess reagents, or unwanted materials from the target surface or sample. The composition of the wash buffer is designed to maintain the integrity and functionality of the equipment or sample during the washing process.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Abcam (2 mentions)
Dapi solution, by Beyotime (1 mentions)
Tcs spe, by Leica (1 mentions)
Alexa fluor 555, by Beyotime (1 mentions)
Anti dystrophin, by Abcam (1 mentions)
Blocking buffer, by Beyotime (1 mentions)
Anti beclin 1, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti vmp1, by Cell Signaling Technology (1 mentions)
Anti ulk1, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated goat anti rabbit igg h l, by Proteintech (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Antibodies to γ h2ax, by Cell Signaling Technology (1 mentions)
Ihc kit, by Abcam (1 mentions)
8 protocols using wash buffer
1
Immunostaining of Dystrophin in Muscle Cells
2
Protein Extraction and Western Blot Analysis
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Protein was extracted from goat LD tissues and primary myoblast cells using a Total Histone Extraction Kit (Beyotime Biotech Co., Ltd., Shanghai, China); protein concentrations were determined using a BCA protein assay kit (Beyotime). The 30 µg of protein per sample was resolved using 12% SDS–PAGE and then transferred onto PVD membranes activated with methanol. After transfer, membranes were blocked with blocking buffer (Beyotime) for 6 h at 4 °C, and then incubated with primary antibodies overnight at 4 °C. The primary antibodies were anti-ULK1 (1:100) (Santa Cruz Biotechnology, New York, NY, USA), anti-VMP1 (1:500), anti-beclin-1 (1:1000), anti-caspase-3 (1:1000), anti-mTOR mammalian (1:1000), and anti-GAPDH (1:1000) (all from Cell Signaling Technology, New York, NY, USA). After incubation, the membranes were rinsed with wash buffer (Beyotime) and incubated for 2 h at 4 °C with secondary antibodies. The secondary Ab was HRP-conjugated goat anti-rabbit IgG (H + L) (Proteintech, Wuhan, China), used at dilutions recommended by Beyotime.
3
Immunofluorescence Staining Protocol
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For immunofluorescence staining, cells were fixed with paraformaldehyde (4%, Beyotime) for 20 min, then permeabilized using Triton X-100 (0.1%, Beyotime) for 20 min and blocked for 1 h in 1% BSA (Beyotime) at room temperature. Then the cells were incubated with indicated primary antibodies (Supplementary Table S1 ) overnight at 4 °C, then cells were washed in wash buffer (Beyotime) four times and incubation with Alexa Fluor 568 (Invitrogen) or Alexa Fluor 488 (Invitrogen) secondary antibodies for 1 h at room temperature, after washing in wash buffer for four times, cells were counterstained with DAPI (Abcam). Cells were imaged with confocal laser microscopy (LSM780, Carl Zeiss).
4
Quantifying DNA Damage in Xenograft Tumors
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Samples from xenograft tumors were fixed using formalin and then used for immunohistochemical staining to measure γH2A.X expression. Tissues were dehydrated in graded ethanol solutions, cleared in 3 changes of xylene, and penetrated in heated paraffin (56–58 °C). The tissues were embedded in paraffin, cut into 4 to 6 mm sections, and placed onto slides. Before staining, deparaffinization and rehydration were performed. Antigen retrieval was performed using a pressure cooker. The slides were incubated in 1× target retrieval solution (Beyotime, Haimen, China) at 120 °C for 4 min at 18 to 20 psi. Endogenous hydrogen peroxidase activity was blocked with hydrogen peroxide for 10 min, followed by rinsing with wash buffer (Beyotime, Haimen, China). The slides were incubated with the appropriate antibodies. The antibodies to γH2A.X were purchased from Cell Signaling Technology, Inc (Danvers, MA, USA). The secondary antibodies against mouse or rabbit IgG were supplied in an IHC kit from Abcam (Cambridge, MA, USA).
5
Ubiquitination of PP2A in Cardiac Tissues
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Cardiac tissues were lysed with lysis buffer containing 20 mM trisHCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, leupeptin, and plus PMSF (Beyotime) on ice for 0.5 h. The cell lysates were centrifuged at 12,000 rpm for 15 min (4 °C) to obtain the protein supernatants. Then, 2 mg/mL of the protein was incubated with anti-PP2A rabbit antibody (0.33 μg) and 30 μL of protein A–Sepharose (Amersham Biosciences), and gently shaken at 4 °C for 24 h. The pellets were washed five times with wash buffer (Beyotime). Bound proteins were eluted with 2× sample buffer with boiling. The ubiquitinated PP2A protein was evaluated with immunoblotting analysis with an anti-ubiquitin (Ub) antibody [4 (link)].
6
Immunofluorescent Staining of Brain Endothelial Cells
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ECs and brain coronal sections were incubated with Ras (1:500; Abcam), Claudin-5, (1:100, Invitrogen), ZO-1 (1:100; Invitrogen), and CD31 (1:50; Invitrogen) antibody overnight at 4 °C. Then, the cells and brain sections were incubated with FITC (green, for Claudin-5 and ZO-1) or Cy3 (red, for CD31) conjugated secondary antibodies (1:250; Invitrogen) for 30 min at room temperature in the dark. Next, the cells and brain sections were washed triple using wash buffer (Beyotime, China), and the cells were incubated with dye for F-actin (rhodamine-phalloidin, 1:1000) for 1 h at room temperature. Cellular nuclear was stained with DAPI (1:1000, Abcam) for 7 min at room temperature. After washing with wash buffer for three times, the fluorescence intensity was detected under a confocal microscope (Leica, TCS SP5II, Germany).
7
NF-κB Phosphorylation Detection Protocol
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Equal amounts of total protein (50 µg) from each lysate were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween 20 and incubated overnight with anti-phospho-NF-κBp65 (Ser536) antibodies (1:1000; Cell Signaling Technology, Danvers, MA, USA) and anti-NF-κBp65 antibodies (1:1000; Abcam, Cambridge, UK) at 4 °C. The membranes were washed thrice with wash buffer (Beyotime Biotechnology, Jiangsu, China) and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:1000; Beyotime Biotechnology, China) for 1 h at room temperature. Lastly, the membranes were subjected to DAB staining to detect the protein bands.
8
Chromatin Immunoprecipitation Assay Protocol
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Chromatin immunoprecipitation (ChIP) assays were performed by using the ChIP assay kit (Beyotime, Shanghai, China) according to the manufacturer's protocols (http://www.beyotime.com/product/P2078. htm). Briefly, GMEC were seeded onto 100 mm × 20 mm cell culture dishes and cultured until 90 to 100% confluence. Then, cells were fixed with 1% formaldehyde for 10 min at 37°C. The cells were washed with cold PBS and cell lysates were sonicated to produce chromatin fragments averaging 200 to 1,000 bp. The fragmented chromatin was then added to ChIP dilution buffer (Beyotime) and the samples were incubated overnight at 4°C with anti-SREBF1 (Abcam, Cambridge, MA) and normal mouse IgG (Millipore, Darmstadt, Germany) antibodies. The resultant immune complexes were precipitated with protein A+G agarose/Salmon Sperm DNA (Beyotime) and washed with wash buffer (Beyotime). Finally, the DNA-protein crosslinks were reversed by incubating the samples in 5 mM NaCl at 65°C for 4 h. The DNA was then treated with proteinase K (Millipore) at 45°C for 1 h and precipitated chromatin was used as the template for PCR with the primers listed in Table 2.
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