For the immunofluorescence assay, PLC-PRF-5 and Hep3B cells subjected to different treatments were washed three times with 1 × PBS, fixed in 4% paraformaldehyde (pre-cooled at 4 °C, Solarbio) for 20 min, and blocked with 5% bovine serum albumin (BSA, KeyGEN BioTECH) containing 0.1% TritonX-100 (Sigma) for 30 min at room temperature. Then, the resultants were incubated with anti-E-cadherin (CST, 14472, 1:50) and anti-Vimentin (CST, 5741, 1:100) or anti-SLUG (Affinity, DF6202, 1:100) and anti-USP5 antibodies (Abcam, ab154170, 1:100). After washing with 1 × PBS again, and then incubated with fluorescently conjugated secondary antibodies (1:200, KeyGEN BioTECH) diluted in 5% BSA for about 50 min at room temperature. Finally, the cells were washed with 1 × PBS and mounted with the DAPI-containing mounting medium (Solarbio). The images of cells were taken with a laser scanning confocal microscope (Leica).
Bovine serum albumin bsa
Manufactured by Keygen Biotech
Sourced in United Kingdom, China
Bovine serum albumin (BSA) is a widely used laboratory reagent derived from bovine blood. It serves as a protein stabilizer, blocker, and carrier in various biological applications.
Automatically generated - may contain errors
Lab products found in correlation
Dapi containing mounting medium, by Solarbio (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Paraformaldehyde (pfa), by Solarbio (3 mentions)
Fluorescently conjugated secondary antibodies, by Keygen Biotech (2 mentions)
Total cell protein extraction kit, by Keygen Biotech (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Ab154170, by Abcam (1 mentions)
Confocal laser scanning microscope, by Leica camera (1 mentions)
Df6202, by Abcam (1 mentions)
Ecl chemiluminescence kit, by Beyotime (1 mentions)
Fluorescent conjugated secondary antibodies, by Keygen Biotech (1 mentions)
Anti stat3, by Affinity Biosciences (1 mentions)
Anti e cadherin, by Affinity Biosciences (1 mentions)
Anti vimentin, by Affinity Biosciences (1 mentions)
Laser scanning confocal microscope, by Nikon (1 mentions)
4 to 20 sds page, by GenScript (1 mentions)
Silver nitrate agno3, by Thermo Fisher Scientific (1 mentions)
Glutaraldehyde ga, by Thermo Fisher Scientific (1 mentions)
Trisodium citrate na3c6h5o7 2h2o, by Sinopharm (1 mentions)
N hydroxysuccinimide, by Merck Group (1 mentions)
6 protocols using bovine serum albumin bsa
1
Immunofluorescence Assay for Cell Markers
2
Protein Extraction and Western Blotting
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Western blotting was performed as previously described [23 (link)]. First, the total protein of GSCs was isolated using the total cell protein extraction kit (KeyGen Biotechnology, Nanjing, China). Then, the proteins were quantified, separated, transferred onto PVDF membranes, blocked with 2% bovine serum albumin (BSA, KeyGen Biotechnology) and incubated with the primary antibodies against PRRX2 or GCH1 (Abcam Technology, Cambridge, UK) at 4 °C overnight. After incubation with secondary antibodies (ProteinTech, Chicago, Illinois, USA), the bands were visualized using a chemiluminescence ECL kit (Beyotime Biotechnology, Beijing, China). All results were quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Immunofluorescence Assay for E-cadherin, Vimentin, YY1, and USP7
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The PLC-PRF-5 and HepG2 cells subjected to different treatments were washed three times with 1× PBS, fixed in 4% paraformaldehyde (precooled at 4 °C, Solarbio) for 20 min, and blocked with 5% bovine serum albumin (BSA; KeyGEN BioTECH) containing 0.1% TritonX-100 (Sigma) for 30 min at room temperature. Then, the cells were incubated with anti-E-cadherin (1:50)/anti-vimentin (1:100) or anti-YY1 (1:100)/anti-USP7 antibodies (1:100). The cells were washed again with 1× PBS and incubated with fluorescent conjugated secondary antibodies (1:200, KeyGEN BioTECH) diluted in 5% BSA for about 50 min at room temperature. Finally, the cells were washed with 1× PBS and mounted with DAPI-containing mounting medium (Solarbio). Cell images were taken with a laser scanning confocal microscope.
4
Immunofluorescence Analysis of Cell Markers
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The PLC/PRF/5 and HepG2 cells with different treatments were washed three times with 1× PBS, fixed in 4% paraformaldehyde (precooled at 4 °C, Solarbio) for 20 min, and blocked with 5% bovine serum albumin (BSA, KeyGen Biotech) containing 0.1% Triton X-100 (Sigma) for 30 min at room temperature. Then, the resultants were incubated with anti-E-cadherin, anti-vimentin, and anti-STAT3 (1:100, Affinity). The cells were washed with 1× PBS again and subsequently incubated with fluorescently conjugated secondary antibodies (1:200, KeyGen Biotech) diluted in 5% BSA for approximately 50 min at room temperature. Finally, the cells were washed with 1× PBS and mounted with the DAPI-containing mounting medium (Solarbio). Images were obtained with a laser scanning confocal microscope (Nikon, Japan).
5
Glioblastoma Stem Cell Protein Extraction and Western Blot Analysis
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Western blotting was performed as previously described [8 (link)]. First, the total cell protein extraction kit (KeyGen Biotechnology, Nanjing, China) was used to isolate the total protein of GSCs. The nuclear and cytoplasmic protein was isolated using a NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Then, the proteins were separated by 4 to 20% SDS-PAGE (Genscript, Nanjing, China), transferred onto polyvinylidene difluoride (PVDF) membranes, and blocked with 2% bovine serum albumin (BSA, KeyGen Biotechnology) and incubated with the primary antibodies at 4 °C overnight, followed with secondary antibodies (ProteinTech, Chicago, Illinois, USA) incubation. The chemiluminescence ECL kit (Beyotime Biotechnology, Beijing, China) visualized the bands. All results were quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
6
Functionalized Silver Nanoparticles for Biosensing
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Polyethyleneimine (PEI), MW 15 000, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. Rose bengal (RB) was purchased from Aladdin Reagent Co., Ltd. Silver nitrate (AgNO3) and glutaraldehyde (GA) were purchased from Alfa Aesar. Trisodium citrate (Na3C6H5O7·2H2O) was purchased from Sinopharm Chemical Reagent Co., Ltd. Human immunoglobulin G (HIgG) and goat anti-human immunoglobulin (GaH-IgG) were purchased from Beijing Bioss Biotech Co., Ltd. Bovine serum albumin (BSA) were purchased from Nanjing KeyGEN Biotech. Co., Ltd. Polyoxyethylene (20) sorbitan monolaurate (Tween-20) and Tris (hydroxymethyl) aminomethane (TRIS) were purchased from Sinopharm Chemical Reagent Co., Ltd. The TBS buffer solution was prepared by dissolving NaCl (8.76 g) and TRIS (1.21 g) in water (500 mL). The TBS-T buffer solution was prepared by mixing Tween-20 (0.25 g) with TBS (500 mL). The BBS solution was prepared by dissolving sodium tetraborate in water to a concentration of 2 mM, reaching a pH of 9.0. P-doped, (100) oriented silicon wafers with a resistivity of 1–10 Ω cm were purchased from Suzhou Ruicai Semi-Conductor Co., Ltd. The water used in the experiments was ultrapure deionized water. All chemical reagents were used as received without further purification.
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