Deparaffinized samples were hydrated, followed by 3% hydrogen peroxide blocking for 15 minutes. The specimens were then immersed in 0.01 M boiling citrate buffer and microwaved for one minute in order to extract antigens. Immediately following blocking with 5% BSA (Boster, Wuhan, China) for 40 minutes, the sections were incubated overnight at 4°C with primary antibodies against AR (1:100), β-catenin (1:100) and HMGB-1 (1:100), respectively. Samples were deparaffinized, hydrated, and blocked with 3% hydrogen peroxide for 15 minutes. Then slices were subjected to antigen retrieval by immersing in 0.01 M boiling citrate buffer and heating in a microwave oven for 1 minute. Sections were blocked by 5% BSA (Boster, Wuhan, China) for 40 minutes at room temperature, and further incubated with primary antibodies against AR (1:100), β-catenin (1:100), and HMGB-1 (1:100) at 4°C overnight, respectively.28 (link) Finally, expression of immunoreactivity was visualized by 3,3’-diaminobenzidine tetrahydrochloride (DAB; Boster, Wuhan, China) after process of incubating with horseradish peroxidase (HRP)-conjugated secondary antibodies (Boster, Wuhan, China) at 37°C for 40 minutes. Using hematoxylin as a nuclear counterstain, the slices were then dehydrated in a graded series of alcohol washes and mounted with coverslips.41 (link)
Bovine serum albumin (bsa)
Manufactured by Boster Bio
Sourced in China, United States
BSA (Bovine Serum Albumin) is a common laboratory reagent used as a protein standard and blocking agent in various biochemical and immunological applications. It is derived from bovine serum and serves as a stabilizer and blocking agent to minimize non-specific binding in assays.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Boster Bio (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Normal goat serum (ngs), by Boster Bio (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (3 mentions)
Dmi6000b, by Leica (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti gapdh antibody, by Abcam (2 mentions)
Western lightning chemiluminescence reagent, by PerkinElmer (2 mentions)
Paraformaldehyde (pfa), by Boster Bio (2 mentions)
Anti ki67, by Thermo Fisher Scientific (2 mentions)
Anti cyp2f2, by Santa Cruz Biotechnology (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Boster Bio (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Boster Bio (1 mentions)
C reactive protein (crp), by Abcam (1 mentions)
Nptx2, by Abcam (1 mentions)
Radioimmunoprecipitation assay (ripa), by Boster Bio (1 mentions)
Cxcr4 antibody, by Boster Bio (1 mentions)
62 protocols using bovine serum albumin (bsa)
1
Immunohistochemical Analysis of AR, β-Catenin, and HMGB-1
2
Immunostaining of NPTX-1, NPTX-2, and CRP in Brain Tissue
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Sections of paraffin-embedded brain slices were deparaffinized and rehydrated before blocking non-specific antigen binding with 5% bovine serum albumin (Boster Biological Technology, China). The following primary antibodies were used: NPTX-1 (1:50; Abcam, Cambridge, UK), NPTX-2 (1:200; Abcam, Cambridge, UK), and CRP (1:100; Abcam, Cambridge, UK). Goat anti-rat/rabbit (Beijing Zhongshan Jinqiao Biotechnology, China) polymer enhancer and goat-resistant rabbit/rat IgG-conjugated polymer were added to each tissue section. Afterward, the immunostained sections were developed in diaminobenzidine (Beijing Zhongshan Jinqiao Biotechnology, China) and counterstained with a weak solution of hematoxylin solution. The stained tissue slices were visualized on a microscope (DMI6000B, Leica, USA).
The immunofluorescence staining protocol on the first day was the same as the immunohistochemical staining protocol, which was performed to evaluate the localization of NPTX-1, NPTX-2, and CRP. The luciferin antibody (100 µL) was added to each stained section. After incubating the sections in the dark at room temperature for 1 h, a drop of 4′,6-diamindino-2-phenylindole (Beijing Zhongshan Jinqiao Biotechnology, China) staining agent was added to each tissue section. An anti-quenching agent was added to seal the sections. The staining results were observed under a fluorescence microscope.
The immunofluorescence staining protocol on the first day was the same as the immunohistochemical staining protocol, which was performed to evaluate the localization of NPTX-1, NPTX-2, and CRP. The luciferin antibody (100 µL) was added to each stained section. After incubating the sections in the dark at room temperature for 1 h, a drop of 4′,6-diamindino-2-phenylindole (Beijing Zhongshan Jinqiao Biotechnology, China) staining agent was added to each tissue section. An anti-quenching agent was added to seal the sections. The staining results were observed under a fluorescence microscope.
3
Quantification of MMP2 and CXCR4 Proteins
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Appropriate amount of RIPA (BOSTER, China) was added to the transfected cells and spinal cord tissues for 3–5 min to completely lyse the cells and tissues, and the supernatant was collected after centrifugation for the determination of total protein using the BCA protein concentration kit (BOSTER, China). The same amount of protein was separated by SDS-PAGE, and the resolved proteins were transferred to a PVDF membrane. The membrane was then blocked with 5% bovine serum albumin (BOSTER, China) for 1.5 hours, and sequentially incubated with primary antibodies overnight at 4°C (MMP2 antibody dilution ratio 1:10000, CXCR4 antibody dilution ratio 1:1000, BOSTER, China), and second antibody (goat anti-rabbit IgG labelled with HRP, dilution 1:5000, absin, China) at room temperature. Western blot analyses were repeated for three times.
4
Immunofluorescence Staining of E-cadherin and Vimentin
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Samples were blocked with goat serum (Boster Biological Technology, Wuhan, China) and incubated with the primary antibody [E-cadherin (1:250), vimentin (1:250)] diluted in 5% bovine serum albumin (BSA; Boster Biological Technology) at 4°C overnight. Secondary antibody conjugated to fluorescein isothiocyanate was used to bind to the primary antibody (1:200) diluted in PBS at 37°C for 30 min. Nuclei were stained with DAPI (1:250; Sigma-Aldrich; Merck KGaA) diluted in PBS at 37°C for 5 min. Samples were placed on glass coverslip. Cells were observed using a confocal laser scanning microscope (Zeiss LSM 510 Meta; Carl Zeiss AG).
5
Quantification and Immunoblot Analysis
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Total protein of samples was extracted by the ice-cold radioimmunoprecipitation lysis buffer (RIPA, Boster, AR0102), which contained protease inhibitor cocktail (Merk, P8340,1:100). We quantified the proteins through the BCA Protein Assay Kit (WSHT, EZPQ01). Subsequently, we separated the protein samples through the SDS-PAGE gels (Servicebio, G2045) and transferred them into the PVDF membranes (ABclonal, RM00018). Then, we blocked the membranes in 5% Bovine serum albumin (Boster, AR0004) and incubated them with primary antibodies: Anti-GAPDH (ABclonal, AC033, 1:1000), Anti-AKT2 antibody (ABclonal, A24009, 1:1000), and Anti-DAPK1 antibody (ABclonal, A5741, 1:1000). The membranes were then incubated by secondary antibodies: Mouse Anti-Rabbit IgG (ABclonal, AS061, 1:1000) and Goat Anti-Mouse IgG (Proteintech, SA00001, 1:1000), followed by enhanced chemiluminescence through the High Sensitivity ECL Kit (Beyotime, P0018S) to display bands.
6
Immunohistochemical Analysis of TLR4 Expression
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Formalin-fixed, paraffin-embedded tissue sections (4 µm) were deparaffinized in xylene, rehydrated through a series of ethanol dilutions, and boiled for 10 min in citrate buffer (10 mM, pH 6.0). Endogenous peroxidase activity was suppressed by exposure to 3% hydrogen peroxide for 10 min. Slides were blocked with 5% bovine serum albumin (Boster, Wuhan, China) and incubated with anti-TLR4 polyclonal antibody (1∶50; Abcam) for 1 h at 37°C. A goat anti-rabbit secondary antibody was used according to the protocol supplied with the ABC Staining System kit (Boster). Sections probed with PBS as primary antibody were considered negative controls. For digital image analysis, the software Adobe Photoshop version 6.0 was used. Results were scored by two independent pathologists as positive, heterogeneous, or negative, when the percentage of stained tumor cells in each section was >75%, between 25% and 75%, and <25%, respectively. The two scores were averaged. The level of staining intensity was recorded as none, weak, moderate, or strong.
7
Immunofluorescence Analysis of Rat Schwann Cells
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RSC96 cells, derived from the long-term culture of rat primary SCs, were purchased from the BaNa Culture Collection of Beijing Beina Chuanglian Biotechnology Institute (Beijing, China). RSC96 cells were cultured using subculturing methods. Dulbecco's modified Eagle's medium/Nutrient Mixture (Boster, Wuhan, China) containing 15% fetal bovine serum (Cellmax, Beijing, China) was replaced every 2–3 days. Cells reaching 80–90% confluency were passaged. Cells were fixed in 4% paraformaldehyde at room temperature for 20–30 minutes, and blocked with 5% bovine serum albumin (Boster) at room temperature for 30 minutes. Cells were incubated with a mouse monoclonal S-100β antibody (1:100; Abcam, Shanghai, China) at 4°C overnight, and then with FITC-labeled goat anti-mouse IgG (1:100; Boster) in the dark at room temperature for 2 hours and then in the dark with DAPI (Boster) for 5 minutes. Cells were examined using fluorescence microscopy (Olympus, Tokyo, Japan) (Chen et al., 2017).
8
Quantifying Oxidative Stress Proteins
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Western blot was used to determine Nrf2, Akt, phosphorylated Akt (p-Akt), and heme oxygenase 1 (HO-1) protein expression. Briefly, total or nuclear protein was extracted using RIPA lysate (Cell Signaling Technology, Boston, MA, USA) or CelLytic™ NuCLEAR™ Extraction Kit (Sigma-Aldrich), respectively, according to the manufacturer's protocol. Protein concentrations were determined with the Bicinchoninic Acid Protein Kit (Cell Signaling Technology). Total proteins (15 μg) of each sample were mixed with 4 μL of 6x loading buffer, loaded on a discontinuous 10% sodium dodecyl sulfate-polyacrylamide gel, separated by electrophoresis, and transferred onto a PVDF membrane (Thermo Fisher Scientific) by the wet method. The membrane was blocked with bovine serum albumin (Boster, Wuhan, China) and skimmed milk for 2 h, washed with tris-buffered saline containing Tween 20 (TBST; Boster), and incubated with the primary antibody rabbit anti-polyclonal antibody IgG (1 : 1000; Abcam, Hong kong, China) at 4°C overnight. After washing with TBST, the membrane was incubated with the horseradish peroxidase-labeled secondary antibody goat anti-rabbit polyclonal antibody IgM (Abcam) at room temperature for 2 h, followed by detection with the enhanced chemiluminescence reagent.
9
Immunofluorescence Staining of Osteogenic Markers
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After the cells were grown on the micropatterned substrates for 3 or 7 days, they were gently washed with PBS, fixed with 4% paraformaldehyde in PBS for 30 minutes at room temperature, and permeabilised with 0.2% Triton X-100 in PBS for 10 minutes at room temperature. The cells were blocked with 5% bovine serum albumin (Boster, Wuhan, China) for 30 minutes, incubated with goat anti-mouse polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) against alkaline phosphatase (ALP, Cat# sc-365765), type I collagen (COL I, Cat# sc-59772), or osteocalcin (OCN, Cat# sc-390877) at a 1:100 dilution for 60 minutes at 37°C, washed with PBS three times, and incubated in the dark for 60 minutes at room temperature with a fluorescein isothiocyanate-labelled affinity-purified antibody to goat IgG (Invitrogen) at a 1:1000 dilution. The staining method used for YAP (A1002, ABclonal, Woburn, MA, USA) and tetraethyl rhodamine isothiocyanate-labelled P-MLC2 (Cat # 3674, Cell Signaling Technology, Boston, MA, USA) was the same as described above. Fluorescence images of these proteins were analysed by ImageJ software. The nuclear/cytoplasmic ratio of YAP in MSCs and MC3T3-E1 cells was obtained by dividing the intensity value in the nucleus to that in the cytoplasm.
10
Western Blot Analysis of Signaling Proteins
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Total cellular protein was extracted with RIPA lysis buffer (Cell Signaling Technology, Boston, MA, USA), and the concentration of total protein was determined by the BCA method (Song et al., 2017). Total protein, 15 μg, was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% bovine serum albumin (Boster, Wuhan, China) at room temperature for 2 hours and immunoblotted overnight at 4°C with primary rabbit anti-rat polyclonal antibodies (1:1000; Abcam) against MKP1, p-JNK (JNK1 + JNK2 + JNK3) and GAPDH, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit polyclonal secondary antibodies (1:4000; Abcam). After extensive washing, protein bands were visualized by an ECL plus chemiluminescence kit (Beyotime Institute of Biotechnology, Haimen, China). Densitometric analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
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