RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitors was utilized to extract the total protein. Protein sample concentration was quantified with the BCA Protein Assay Kit (Takara, Shanghai, China). The extracted proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electro-transferred to a polyvinylidene fluoride membrane (Takara). After blocking with 5% bovine serum albumin for 2 h at room temperature, the membranes were incubated at 4°C with primary antibodies diluted with primary antibody dilution buffer (Solarbio) as follows: CRIM1 (1:500 dilution; Abcam, Hong-Kong, China), E-cadherin (1:100 dilution; Abcam), vimentin (1:1500 dilution; Abcam), N-cadherin (1:1500 dilution; Abcam), and β-actin (1:5000 dilution; Abcam). After incubation with peroxidase-conjugated secondary antibodies (goat anti-rabbit, 1:5000 dilution; Proteintech, Chicago, IL, USA) at room temperature, the signal was detected using ECL chemiluminescent reagents (Pierce, Rockford, IL, USA). The abundance of target protein bands was measured and analyzed using the chemiluminescence Western blotting detection system (Tanon, Shanghai, China).
Primary antibody dilution buffer
Manufactured by Solarbio
Sourced in China, United States
Primary Antibody Dilution Buffer is a ready-to-use solution designed to dilute primary antibodies. It helps maintain the stability and specificity of primary antibodies during immunoassay procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ecl luminescence reagent, by Solarbio (3 mentions)
E cadherin, by Abcam (2 mentions)
N cadherin, by Abcam (2 mentions)
β actin, by Abcam (2 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Takara Bio (1 mentions)
Vimentin, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Takara Bio (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Application suite, by Leica (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Glut1, by Proteintech (1 mentions)
Ix71 fluorescence microscope, by Olympus (1 mentions)
Ve cadherin, by Santa Cruz Biotechnology (1 mentions)
P glycoprotein, by Cell Signaling Technology (1 mentions)
Ccd digital camera, by Leica (1 mentions)
Blocking serum, by Solarbio (1 mentions)
8 protocols using primary antibody dilution buffer
1
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
2
Western Blot Protein Detection
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Proteins were parted by electrophoresis on 8% or 10% SDS-PAGE gels and then transferred onto 0.45 μm PVDF membranes. The membranes were then obstructed with non-fat milk for 1–2 h at room temperature. All antibodies were diluted in Primary Antibody Dilution Buffer (Solarbio, Cat. No. A1810) at 1:1000. Finally, protein bands were perceived on X-ray film through ECL luminescence reagent (Solarbio, Cat. No. SW2010).
3
Immunostaining of BBB Markers
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Cell samples were washed three times in PBS before fixing in 4% paraformaldehyde (Sigma) for 30 min and permeabilized in 0.2% Triton X-100 solution (Sigma) for 15 min. The samples were then blocked in blocking serum (Solarbio) and incubated with VE-Cadherin (Santa Cruz), ZO-1 (Santa Cruz), Claunin-5 (Proteintech), P-glycoprotein (Cell Signaling), Glut-1 (Proteintech), respectively diluted in primary antibody dilution buffer (Solarbio) at 4 °C for 24 h. Target proteins of the samples were detected by incubating cells in fluorophore-conjugated secondary antibodies (Zhongshan Golden Bridge) for 1 h at room temperature away from light. Cell nuclei were counterstained with DAPI (Sigma). Images were obtained with an Olympus IX71 fluorescence microscope (Olympus Corporation) equipped with a CCD digital camera (Leica) and processed with Leica Application Suite (Leica) and Image-Pro Plus software (Media Cybernetics, USA). Fluorescence images of single BBB region were taken using a Laser Scanning Confocal Microscope (Olympus Fluo View TM FV1000).
4
Immunohistochemical Analysis of Apoptosis Markers
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Tissue sections were blocked using 5% Donkey serum (cat. no. SL050; Beijing Solarbio Science & Technology Co., Ltd.; diluted in PBS) for 30 min at 4˚C, and incubated with antibodies against caspase 3 (1:100), PARP-1 (1:100), or β-actin (1:200) overnight at 4˚C, the antibodies were diluted using Primary Antibody Dilution Buffer (cat. no. AB64211; Abcam). The slides were washed 3 times with PBS, and incubated with the corresponding secondary antibody (1:500; cat. no. A0208; Beyotime Institute of Biotechnology) and treated with a streptavidin derivative coupled to alkaline phosphatase (cat. no. 21324; Thermo Fisher Scientific, Inc.). The sections were stained with DAB chromogen A for 5 min at room temperature (cat. no. P0203; Beyotime Institute of Biotechnology). Subsequently, the slides were examined at x20 magnification using an Olympus BX-UCB light microscope.
5
Western Blot for CD73 Protein
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The cell total protein extraction and western blotting were performed as previously described [14 (link)]. To be specific, the total protein was extracted from cells by RIPA buffer (Solarbio, Beijing, China) supplemented with Protease Inhibitor Cocktail (Bimake, USA). Protein samples were mixed with 4 × loading buffer (Solarbio, Beijing, China) and boiled for 5 min at 95 °C. Next, 30 μg of total protein was separated with 10% SDS-PAGE gel. After blocked with 5% nonfat milk, membranes were incubated overnight at 4 ℃ with the anti-CD73 (clone EPR6114, dilution at 1:1000, Abcam, USA) antibodies diluted in Primary Antibody Dilution Buffer (Solarbio, Beijing, China). Then, they were washed with TBST, and the membranes were incubated for 60 min with horseradish peroxidase-linked goat-anti-rabbit secondary antibody (dilution at 1:2000, Cell Signaling Technology, USA). Finally, membranes were developed with the electro-chemiluminescence solution (Millipore, USA), and images were taken on a ChemiScope exposure machine (Clinx Science Instruments Co, Ltd, Shanghai, China).
6
Western Blot and Immunohistochemistry Protocols
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Proteins were parted by electrophoresis on 8% or 10% SDS-PAGE gels and then transferred onto 0.45 μm PVDF membranes. The membranes were then obstructed with non-fat milk for 1-2 hours at room temperature. All antibodies were diluted in Primary Antibody Dilution Buffer (Solarbio, Cat. No. A1810) at 1:1000. Finally, protein bands were perceived on X-ray lm through ECL luminescence reagent (Solarbio, Cat. No. SW2010 ).
Immunohistochemistry analysis IHC analysis of YAP1 was executed through the Dako Envision TM FLEX + System (Dako, Glostrup, Denmark) as explained previously [21] .
Immunohistochemistry analysis IHC analysis of YAP1 was executed through the Dako Envision TM FLEX + System (Dako, Glostrup, Denmark) as explained previously [21] .
7
Western Blot Protein Detection
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Proteins were parted by electrophoresis on 8% or 10% SDS-PAGE gels and then transferred onto 0.45 μm PVDF membranes. The membranes were then obstructed with non-fat milk for 1-2 hours at room temperature. All antibodies were diluted in Primary Antibody Dilution Buffer (Solarbio, Cat. No. A1810) at 1:1000. Finally, protein bands were perceived on X-ray film through ECL luminescence reagent (Solarbio, Cat. No. SW2010 ).
8
Protein Expression Analysis by Western Blot
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Cells were collected and lysed in RIPA lysis buffer (Solarbio) on ice, then extracted proteins were quantified and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Solarbio). The PVDF membranes were blocked using 5% skimmed milk for 2 h with gentle shaking, then incubated with primary antibodies diluted with primary antibody dilution buffer (Solarbio) as follows: E-cadherin (1:100; Abcam, Cambridge, MA, USA), N-cadherin (1:2000; Abcam), SLUG (1:2000; Abcam), MMP9 (1:2000; Abcam), OCT1 (1:1500; Abcam) and β-actin (1:2500; Abcam). After incubation at 4 °C overnight, the membranes were thoroughly washed and incubated with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit, 1:5000; Wanleibio, Shenyang, Liaoning, China) at room temperature for 2 h. We utilized a chemiluminescence western blotting detection system (Tanon, Shanghai, China) to analyze the protein bands.
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