ACHN and 786-O cells were fixed with 4% paraformaldehyde for 15 min at room temperature, then washed with PBS 3 times and permeabilized with 0.3% Triton X-100 for 20 min at room temperature. Samples were blocked with 3% BSA (Sangon Biotech Co., Ltd.) for 30 min at room temperature and incubated with specific primary antibodies overnight at 4°C in 3% BSA. After washing, the fluorescently labelled secondary antibodies were incubated for 1 h at room temperature. After further washing, nuclei were stained with DAPI at room temperature for 10 min. Slides were observed and imaged using the EVOS FL Auto 2.0 Imaging System (Thermo Fisher Scientific, Inc.).
Bovine serum albumin (bsa)
Manufactured by Sangon
Sourced in China, United States, Germany
Bovine Serum Albumin (BSA) is a common laboratory reagent used in various biological and biochemical applications. It serves as a protein source, stabilizer, and blocking agent. BSA is widely utilized in assays, cell culture media, and buffer solutions to maintain the integrity and functionality of proteins and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (12 mentions)
Triton x 100, by Merck Group (10 mentions)
Ripa lysis buffer, by Beyotime (9 mentions)
Ripa buffer, by Beyotime (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (7 mentions)
Triton x 100, by Beyotime (7 mentions)
Hematoxylin, by Solarbio (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Polyvinylidene difluoride membrane, by Merck Group (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Goat anti rabbit hrp conjugate, by Cell Signaling Technology (5 mentions)
Tween 20, by Sangon (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Paraformaldehyde (pfa), by Sangon (4 mentions)
Fluorescence microscope, by Olympus (4 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Lsm 710 confocal microscope, by Zeiss (4 mentions)
Triton x 100, by Sangon (3 mentions)
263 protocols using bovine serum albumin (bsa)
1
Immunofluorescence of Renal Cancer Cells
2
Bovine Serum Albumin-Loaded Calcium Phosphate and Hydroxyapatite Coatings
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A protein of bovine serum albumin (BSA, Sangon Biotech Ltd., China) has been chosen to prepare the BSA-containing ACP-PLA and BSA-containing HA-PLA suspensions for coating Ta plates (1 cm × 1 cm × 0.1 mm). The BSA-containing ACP nanospheres and HA nanorods were prepared according to the modified method used in the preparation of growth factors-containing ACP-PLA and HA-PLA modified Ta scaffolds. The 30 mg of ACP or HA were separately dispersed in BSA solution (5 mL, 1 mg mL−1). The suspension was shaken at a constant rate of 130 rpm in a sealed vessel at 37 °C for 2 h, followed by freeze-drying to obtain the BSA-loading ACP nanospheres and HA nanorods. The coating process is similar to above preparation process of ACP-PLA and HA-PLA modified Ta scaffolds.
The in vitro release experiments were performed as follows: the BSA-loading Ta plates were immersed into SBF (80 mL) at 37 °C under shaking at a rate of 160 rpm. The BSA release medium (2 mL) was withdrawn for the UV-vis analysis at 562 nm using the Bicinchoninic Acid Kit (BCA, Beyotime Institute of Biotechnology, China) at given time intervals. The withdrawn solution was replaced with fresh SBF (2 mL, 37 °C). Thereafter, the release curves were obtained and used for the analysis of in vitroBSA release.
The in vitro release experiments were performed as follows: the BSA-loading Ta plates were immersed into SBF (80 mL) at 37 °C under shaking at a rate of 160 rpm. The BSA release medium (2 mL) was withdrawn for the UV-vis analysis at 562 nm using the Bicinchoninic Acid Kit (BCA, Beyotime Institute of Biotechnology, China) at given time intervals. The withdrawn solution was replaced with fresh SBF (2 mL, 37 °C). Thereafter, the release curves were obtained and used for the analysis of in vitroBSA release.
3
Evaluating T. gondii Antibody Titers and Cytokine Profiles
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To investigate the titers of T. gondii-specific serum antibody in the sera, ELISAs were conducted as described previously (44 (link)). Briefly, each well (96-well plates, Costar, Cambridge, MA, USA) was coated with 1 μg of STAg (dissolved in 100 μl of carbonate buffer pH 9.6) overnight at 4°C. After 5-min rinsing in TBST, each well was blocked with TBST containing 5% BSA (Sangon Biotech, Shanghai, China) at 37°C for 1 h. Sera from mice were diluted (1:100) in TBST containing 5% BSA and added into each well after being rinsed in TBST for 5 min. Incubated at 37°C for 1 h, each well of the 96-well plates was rinsed 5 min in TBST and incubated with HRP-conjugated anti-mouse IgG, IgG1, or IgG2a (1:8,000, eBioscience, San Diego, USA) at 37°C for 1 h. Finally, tetramethylbenzidine (TMB; Tiangen, Beijing, China) as substrate was used to evaluate the immunoreaction, and the reaction in each well was stopped by 100 μl of 2 M newly prepared H2SO4. The absorbance was determined at 450 nm by a microplate reader (Thermo Scientific, Waltham, MA, USA).
To determine cytokine secretions in animals’ sera, commercially available ELISA kits (Yifeixue Biotech, Nanjing, China) based on double antibody sandwich method were used to evaluate the concentrations of interferon-gamma (IFN-γ), interleukin (IL) 4 (IL-4), IL-10, and IL-17 referenced to known amounts of mouse recombinant cytokines.
To determine cytokine secretions in animals’ sera, commercially available ELISA kits (Yifeixue Biotech, Nanjing, China) based on double antibody sandwich method were used to evaluate the concentrations of interferon-gamma (IFN-γ), interleukin (IL) 4 (IL-4), IL-10, and IL-17 referenced to known amounts of mouse recombinant cytokines.
4
PITX3 Subcellular Localization Assay
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HeLa cells were plated onto coverslips in 12-well plates and seeded at 2×104 cells per well in DMEM with 10% FBS for 24 h. Cells were transfected with 1.5 µg of PITX3 wild-type and mutants plasmids respectively using transfection reagent Vigofect (Vigorous Biotechnology, Inc., Beijing, China) and harvested 24 h post-transfection. According to the manufacturer's protocol. The cells were washed with PBS 3 times, fixed with purity methanol (100% methanol) at −20°C for 10 min, permeabilized with 0.25% Triton X-100 at 25°C for 10 min, blocked with 1% bovine serum albumin (Sangon Biotech Co., Ltd., Shanghai, China) at 25°C for 1 h, and incubated with a primary antibody PITX3 (N-20) (cat. no. sc-19307; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at a 1:1,000 dilution at 25°C for 1 h. Following washing 3 times with PBS, the cells were subsequently incubated with secondary antibody Alexa Fluor 594 Donkey anti-Goat IgG at a 1:200 dilution (cat. no. A11058; Thermo Fisher Scientific, Inc.) for 1 h at 25°C. Nuclei were counterstained with DAPI dye (0.5 µg/m1) for 1 min at 25°C. Finally, the location of PITX3 wild-type and mutants protein was detected by confocal fluorescent microscopy at two different magnifications: ×20 and ×40.
5
Histological Analysis of Mouse Articular Cartilage
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The mouse cartilage tissues were decalcified in 10% disodium ethylene diamine tetraacetic acid (Biosharp, Hefei, China), embedded in paraffin and cut into 5-µm-thick sections. All tissues were stained with safranin O-fast green. The articular cartilage destruction was evaluated using the Osteoarthritis Research Society International (OARSI) scoring system by estimating the highest observed scores based on a previous study (Glasson et al. 2010 (link)). For immunohistochemistry (IHC), the sections embedded in paraffin were dewaxed, hydrated, and inoculated with 3% hydrogen peroxide. Thereafter, the sections were blocked with 1% bovine serum albumin (Sangon, Shanghai, China) for 15 min at room temperature and incubated with the primary antibodies against CHMP5 (1:50), collagen II (1:50) and matrix metallopeptidase 13 (MMP13; 1:50) purchased from Affinity Biosciences (Changzhou, China) at 4 °C overnight. Then, the slides were inoculated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:500; Thermo Fisher Scientific, Shanghai, China) at 37 °C for 1 h. Next, the 3, 3-diaminobenzidine tetrahydrochloride (Maixin, Fuzhou, China) was used as a chromogenic agent. The images were captured with a microscope (Olympus, Japan).
6
Quantifying Protein Levels via Western Blot
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Radioimmunoprecipitation assay (RIPA) solution was utilized to extract the total protein of the cells and BCA reagent (Beyotime, Shanghai, China) was used to determine their concentration. Protein samples were separated by electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore, USA) for 2 h. Subsequently, the membranes were blocked with 3% bovine serum albumin (Sangon, Shanghai, China) and incubated with anti-WDR72 primary antibody (Cell Signaling, Danvers, MA, USA) at 4° C overnight. The next day, 1×TBST was used to wash the membranes 3 times. Then, a secondary antibody was used to incubate the membranes at room temperature for 1.5 h. Finally, the bands were scanned by a gel-imaging platform (UVP, CA, USA).
7
Visualizing Phosphorylated NF-κB Localization
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Transparent glass slides were placed at the bottom of six-well plates, coated with poly-D-lysine solution (Sangon Biotech, Shanghai, China) for 30 min, rinsed with phosphate-buffered saline, and dried. Cells (2 × 105 per well) were added and cultured with medium containing LPS for 6 h. The medium was aspirated, and cells were washed with PBS and fixed with 4% paraformaldehyde (Sangon Biotech, Shanghai, China) before being washed three more times with PBS and incubated with 0.1% Triton X-100 (Sangon Biotech, Shanghai, China) for 15 min. After blocking with 3% bovine serum albumin (Sangon Biotech, Shanghai, China) for 1 h, cells were incubated with phospho-p65 antibody (#3033, Cell Signaling Technology, Danvers, MA, USA) for 1 h, washed three times, and incubated with Alexa Fluor 488 (Abcam, Cambridge, UK) for 1 h. Cells were then observed under a laser confocal microscope (Olympus FV1000, Tokyo, Japan).
8
Western Blot Analysis of NF-κB Pathway
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HSG cells were washed with PBS and lysed with RIPA buffer (Beyotime). The cell lysates were separated on 10% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 3% bovine serum albumin (Sangon) in TBS for 2 h and then incubated with primary antibodies against GAPDH (AF1186, Beyotime), PIGR (A6130, Abclonal), NF-κB (8242S, Cell Signaling Technology, Beverly, MA, USA), and phospho-NF-κB (3033, Cell Signaling Technology) at 1:1,000, overnight at 4°C. Primary antibodies were labeled by incubation with secondary antibodies (1:10,000, 5151s, Cell Signaling Technology) at 20°C for 1.5 h. Antibody-bound proteins were detected using an Odyssey® CLx Imaging System (LI-COR, Lincoln, NE, USA). GAPDH antibody levels were used for normalization.
9
Measuring IL-33 Expression in Synovial Fibroblasts
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The synovial fibroblasts were put on cover slides in the 6-well plate and were treated with MSU, TNFα, or IL-1β for 24 h. Next, the cell slides were taken out for immunohistochemistry analysis. Cell slides were fixed for 5 min in 4% paraformaldehyde solution, rinsed twice with PBS, followed by 0.5% Triton-100 solution treatment. The slides were treated with 3% BSA (Sangon Biotech, Shanghai) for 30 min at 37°C for blocking non-specific staining. After that, the slides were then incubated with goat anti-human IL-33 Ab (Minneapolis, MN, United States) or control gout IgG at 4° overnight, and were then used hypersensitive two-step immunohistochemical detection reagent (ZSGB-BIO, China) to detect the IL-33 expression levels by microscope.
10
Immunofluorescence Staining Protocol
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The cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, Germany), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 10 min, washed 3 times for 5 min/wash with PBS, and then blocked with 3% BSA (Sangon Biotech, China) in PBS (Sangon Biotech, China) for 1 h at room temperature. The cells were incubated with the primary antibodies (Supplementary Table S2 ) and then incubated overnight at 4°C. Then, they were washed 3 times for 5 min/wash with PBS and incubated with the following fluorescent secondary antibodies for 1 h at room temperature: Alexa Fluor 555-conjugated donkey anti-mouse (1:500, Thermofisher, United States) and Alexa Fluor 488-conjugated donkey anti-mouse (1:500, ThermoFisher, United States). After three washes for at least 10 min each, the cells were exposed to DAPI (Sigma-Aldrich, Germany) for 5 min at room temperature to visualize nuclei. The samples were washed 3 times for 5 min each, and then pictures were taken with a Olympus microscope (Olympus, Japan).
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