Brain tissue samples were harvested 2 weeks after NSC transplantation. Tissues were lysed in cold lysis buffer (1X PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% SDS; RIPA) containing proteinase inhibitors. The protein concentration was measured using a bicinchoninic protein assay kit. An equal amount of protein samples (50 µg) was loaded in each lane. The protein samples (concentration, 5 µg/µl; volume, 10 µl) were submitted to 10% SDS-PAGE prior to electrotransfer on a PVDF membrane. The membrane was incubated in 5% non-fat milk at room temperature for 1 h and washed three times with TBST (0.01 M TBS and 0.1% Tween-20). The specific primary antibodies: Rabbit anti-ChAT (1:1,000), rabbit anti-Map2 (1:1,000), rabbit anti-PSD95 (1:1,000), rabbit anti-SYP (1:1,000), mouse anti-β-tubulin III (1:1,000) were incubated with the membrane overnight at 4°C. After washing in TBST to remove residual antibodies, horseradish peroxidase (HRP) conjugated secondary antibodies (goat anti-rabbit IgG; 1:2,000 and rabbit anti-mouse 1:1,000) were applied for 2 h at room temperature. The membrane was then washed with TBST prior to film exposition and development using an ECL kit (Bioworld Technology, Inc.). The protein expression levels were analyzed using ImageJ (version 1.4.3.67; National Institutes of Health).
Ecl kit
Manufactured by Bioworld Technology
Sourced in United States
The ECL kit is a laboratory equipment used for chemiluminescent detection of proteins on Western blots. It provides the necessary reagents to enable the visualization and analysis of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (3 mentions)
Bca protein assay kit, by Bioworld Technology (2 mentions)
Bca kit, by Beyotime (2 mentions)
Quantity one v4, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti ide, by Abcam (1 mentions)
Anti psd95, by Cell Signaling Technology (1 mentions)
Anti syn, by Abcam (1 mentions)
Anti app, by Abcam (1 mentions)
Anti ps1, by Cell Signaling Technology (1 mentions)
Protein extraction kit, by Beyotime (1 mentions)
Imagej software, by Bio-Rad (1 mentions)
Anti claudin 5, by Abcam (1 mentions)
Anti gapdh, by Bioworld Technology (1 mentions)
Anti occludin, by Thermo Fisher Scientific (1 mentions)
Anti cd31, by Abcam (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Stat5, by Cell Signaling Technology (1 mentions)
Phospho stat5, by Cell Signaling Technology (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
12 protocols using ecl kit
1
Western Blot Analysis of Neural Markers
2
Western Blot Analysis of Hippocampal Proteins
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Hippocampus were rinsed twice with cold PBS and dissolved on ice with a RIPA buffer containing 1 Mm Phenylmethanesulfonyl fluoride (Promega Corporation) for 30 min, followed by centrifugation at 12,000 x g for 10 min at 4˚C. Protein concentrations were determinate using the BCA protein assay. Total proteins (50 µg/well) were separated via 10% SDS-PAGE and transferred via electrophoresis onto PVDF membranes. Membranes were blocked with 5% skimmed milk at room temperature for 1 h and incubated overnight at 4˚C with anti-PKA (1:1,500), anti-CREB (1:600), anti-phosphorylated-CREB (p-CREB; 1:1,000), anti-BDNF (1:1,200) and anti-actin (1:3,000). Actin was used as the loading control. Subsequently, membranes were incubated with corresponding secondary antibodies (1:1,000) at 37˚C for 2 h and reactions were visualized with chemiluminescence reagents provided by an ECL kit (Bioworld Technology, Inc.) and exposed to X-ray film. Blot intensities were quantified with densitometry by Quantity One v4.6.6 (Bio-Rad Laboratories).
3
Quantifying Cellular Proteins by Western Blot
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Total proteins in cells were prepared using RIPA and separated using 8% SDS-PAGE. After separation, proteins were transferred onto PVDF membranes (Millipore). After blocking, the PVDF membrane was incubated with SERBP1 or GAPDH antibody for overnight. After incubating with HRP-conjugated secondary antibody, the target bands were measured by using an ECL kit (Bioworld).
4
Proteomic Analysis of Hippocampal Proteins
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Proteins were isolated from the hippocampus of brain according to the protein extraction kit instructions (Beyotime Biotechnology, China). Protein concentrations were determined using the BCA protein assay kit (Bioworld, USA). Equal volumes of protein were separated using 10% dodecyl sulfate, sodium salt polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 5% non-fat milk for 1 h and incubated overnight at 4 °C with primary antibodies as follows: 6E10 (1:1000; Covance, USA), anti-APP (1:1000, Abcam Technology, UK), anti-PSD95 (1:1000, Cell Signaling Technology, USA), anti-SYN (1:1000, Abcam Technology, UK), anti-IDE (1:1000,Abcam Technology, UK), anti-ADAM10 (1:1000, Millipore Technology, USA), anti-BACE1 (1:1000, Millipore Technology, USA), anti-PS1 (1:1000, Cell Signaling Technology, USA), and anti-GRPDH (1:2000, Cell Signaling Technology, USA). Membranes were rinsed with Tris-buffered saline with Tween 20 (TBST) three times. Then, membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies for 2 h. Protein signals were visualized with the chemiluminescence reagents provided with the ECL kit (Bioworld, USA), and quantitation of proteins was determined by densitometric analysis using ImageJ software (Bio-Rad, Hercules, USA).
5
Quantifying Protein Expression in Mice Brain
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Mice were euthanized and decapitated, and their striatum was quickly dissected on ice. Brain samples and treated astrocytes in vitro were homogenized in RIPA buffer (Beyotime Biotechnology) containing a mixture of phosphatase and protease inhibitors. The protein concentration was then determined using a BCA kit (Beyotime Biotechnology). Western bolt was executed according to a standard protocol. The hydrophilic polyvinylidene fluoride (PVDF) membrane, which was blocked in 5% skim milk powder for 1 hour at room temperature, was incubated in anti‐GAPDH (1:5000, Bioworld), anti‐claudin‐5 (1:500, Abcam), anti‐occludin (1:500, Invitrogen), anti‐CD31 (1:500, Abcam), anti‐MPO (1:500, CST), anti‐MMP‐2 (1:500, CST) and anti‐MMP‐9 (1:500, CST) antibodies overnight at 4°C. After washing with TBS buffer, the membrane was incubated with the appropriate HRP‐conjugated secondary antibody for 2 hours at room temperature. The immunoreactive bands were visualized with a chemiluminescent reagent provided with an ECL kit (Bioworld). The blot strength was detected using ImageJ software.
6
Protein Expression Analysis by Western Blot
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Western blot was performed as previously described. Equal amounts of proteins were separated by sodium dodecyl sulfate-PAGE electrophoresis and then blotted onto polyvinylidene fluoride membranes. After being blocked in 5% fat-free milk for 2 hours, membranes were incubated with primary antibodies against TLR2 (1 : 1000, Abcam, UK), TLR3 (1 : 1000, Abcam, UK), TLR6 (1 : 1000, Cell Signaling Technology, USA), MAPK p38 (1 : 500, Cell Signaling Technology, USA), phospho-p38 (1 : 500, Cell Signaling Technology, USA), CD36 (1 : 1000, Abcam, UK), SYK (1 : 1000, Abcam, UK), STAT3 (1 : 500, Cell Signaling Technology, USA), phospho-STAT3 (1 : 500, Cell Signaling Technology, USA), STAT5 (1 : 500, Cell Signaling Technology, USA), phospho-STAT5 (1 : 500, Cell Signaling Technology, USA), and GAPDH (1 : 5000, Bioworld, USA) in 4°C overnight to probe targeted proteins. Horseradish peroxidase-conjugated secondary antibodies (1 : 5000, Bioworld, USA) were used to combine primary antibodies and the reaction was detected with an ECL Kit (Bioworld, USA). The intensities of blots were quantified by densitometry.
7
Quantitative Western Blot Analysis
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The proteins were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% skimmed milk for 1 h and incubated overnight at 4 °C with anti-actin and anti-HMGB1. Actin was used as a loading control. Subsequently, the membranes were incubated with the corresponding secondary antibodies, and the reaction was visualized with chemiluminescence reagents provided with an ECL kit (Bioworld) and exposed to X-ray film. The intensity of the blots was quantified with densitometry.
8
Hippocampus Protein Extraction and Analysis
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Three animals from each group were euthanized and decapitated, and the hippocampus of the mice were removed and sonicated in RIPA buffer (Beyotime Institute of Biotechnology, China) containing phosphatase and protease inhibitor cocktails (Sigma-Aldrich, USA). The total protein was obtained from the supernatant of this extract following 30 min of centrifugation at 12000×rpm, and the protein content was quantified by the BCA protein assay kit (Bioworld, USA). Western blotting was performed as described previously[23 (link)]. Samples (30 μg protein) were separated by SDS-PAGE and electrophoretically transferred onto PVDF membranes. Membranes were blocked for 1 h in 5% non-fat milk and incubated overnight with primary antibodies. β-actin was used as a loading control. The incubation with primary antibodies was followed by a 2 h incubation with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies. The protein signals were detected using the chemiluminescence reagents provided with the ECL kit (Bioworld, USA), and Image J software was used to determine the intensity of the blots.
9
Molecular Markers of Ischemic Brain Injury
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The rats were euthanized and decapitated, and their ischemic ipsilateral cortices were rapidly dissected on ice and stored at −80 °C until needed. The samples were then taken from the freezer and sonicated in RIPA buffer (Beyotime Institute of Biotechnology, China) containing phosphatase and protease inhibitor cocktails. A BCA Kit (Beyotime Biotechnology) was then used to determine the protein concentrations. Equal amounts of sample were separated by SDS-PAGE and transferred onto PVDF membranes, which were blocked in 5% non-fat dry milk at room temperature for 1 h. The membranes were then incubated overnight at 4 °C with primary antibodies against VEGF (1:1000 Bioworld Technology, USA), Ang-1 (1:1000 Bioworld Technology, USA), Shh (1:250 Life Technology, USA), Patched-1 (1:1000 Abcam, UK), Smoothened (1:1000 Abcam, UK) and Beta-actin (1:5000 Bioworld, USA). The next day, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies for 2 h at room temperature. Immunoreactive bands were visualized with chemiluminescence reagents that were provided with an ECL kit (Bioworld, USA). Blot intensity was detected using Image J software.
10
Protein Expression Analysis by Western Blot
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Equal quantities of protein extracts were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, USA). After blocking in 5% non-fat milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: MFG-E8 (Santa Cruze), NG2 (Millipore), and PLP1 (Abclone). The membranes were subsequently incubated with corresponding secondary antibodies and visualized with chemiluminescence reagents provided with an ECL kit (Bioworld, USA).
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