Different concentrations of COF samples were added to 4T1 cells and incubated overnight. Following various treatments, the 4T1 cells (2 × 103) were fixed with 4% paraformaldehyde, infiltrated with 0.5% Triton X-100 (BS084, Biosharp), blocked with 3% BSA (Biosharp BS114) in 0.1% triton/PBS for 1 hour, and incubated with anti-HMGB1 antibodies (1:400, ab18256, Abcam) at room temperature and Goat anti-Rabbit IgG DyLight 488 (1:200, A23220, Abbkine). The cells were then counterstained with DAPI (Beyotime Biotechnology, P0131), The slides were examined with an FV1000 confocal microscope (Olympus) using FV10-ASW software v. 4.0 (Olympus). The fluorescence signals were analyzed using Image-Pro Plus software v. 6.0 (Media Cybernetics).
Bs084
Manufactured by Biosharp
Sourced in China
The BS084 is a centrifuge designed for general laboratory use. It features a brushless DC motor and digital speed control for efficient and accurate operation. The centrifuge has a maximum speed of 6,000 RPM and can accommodate various rotor configurations to suit different sample volumes and types.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Ab18256, by Abcam (1 mentions)
Image pro plus software v 6.0, by Media Cybernetics (1 mentions)
Bs114, by Biosharp (1 mentions)
Dylight 488 goat anti rabbit igg, by Abbkine (1 mentions)
A23220, by Abbkine (1 mentions)
Fv10 asw software v 4, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Ab122333, by Abcam (1 mentions)
Ab122367, by Abcam (1 mentions)
T6793, by Merck Group (1 mentions)
Hpa021624, by Merck Group (1 mentions)
Nbp2 14260, by Novus Biologicals (1 mentions)
Alexa fluor 488, by Zeiss (1 mentions)
Alexa fluor 594, by Zeiss (1 mentions)
Hpa038440, by Merck Group (1 mentions)
Hpa017382, by Merck Group (1 mentions)
Hpa020046, by Merck Group (1 mentions)
Hoechst 33342, by Zeiss (1 mentions)
5 protocols using bs084
1
Quantifying HMGB1 in 4T1 Cells
2
Sperm Flagellar and Acrosomal Protein Analysis
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A sperm sample from patient AY0283 and two normal sperm samples fixed with 4% PFA were applied to slides, permeabilized with 0.1% Triton X-100 (BS084, Biosharp) for 10 min, and then blocked with 10% donkey serum for 2 h at 37°C. To analyze the location and expression of DNAH6 in spermatozoa and the changes in flagellar-associated and acrosome-associated proteins, anti-DNAH6 (rabbit, 1:100, ab122333, Abcam, Cambridge, UK), anti-DNAH1 (rabbit, 1:100, ab122367, Abcam), anti-DNAI2 (rabbit, 1:200, 17533-1-AP, Proteintech), anti-SPAG6 (rabbit, 1:200, HPA038440, Sigma-Aldrich), anti-RSPH1 (rabbit, 1:100, HPA017382, Sigma-Aldrich), and anti-AKAP4 (rabbit, 1:200, HPA020046, Sigma-Aldrich) antibody were co-incubated with monoclonal anti-acetylated-tubulin antibody (mouse, 1:500, T6793, Sigma-Aldrich) at 4°C for 16 h. Anti-ACTL7A (rabbit, 1:100, HPA021624, Sigma-Aldrich) and anti-acrosin antibodies (rabbit, 1:200, NBP2-14260, Novus) were also incubated separately at 4°C for 16 h. We used Alexa Fluor 488 anti-mouse antibody (1:800, Jackson, Lancaster, PA, USA), Alexa Fluor 594 anti-rabbit antibody (1:800, Jackson), and Hoechst 33 342 (1:500, Thermo Scientific) as secondary antibodies. The stained samples were observed with a laser scanning confocal microscope (LSM800, Carl Zeiss AG) in selected channels (Alexa Fluor 594, Alexa Fluor 488, and Hoechst 33 342).
3
Immunofluorescence Staining of Cellular Markers
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EPCs (5 × 104 cells) were seeded on sterilized coverslips. After treatment, cells were washed three times with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) and fixed by 4% paraformaldehyde (PFA, C104188, ALADDIN, Shanghai, China) for 30 min. The cell membrane was perforated by 0.3% Triton X-100 (Biosharp, BS084, Hefei, China) for 15 min, and the cell was blocked with 5% bovine serum albumin (BSA, Solarbio, A8010, Beijing, China) for 1.5 h. The primary antibodies rabbit anti-NLRP3 (1:200, Abways Technology, Inc., CY5651, Shanghai, China), rabbit anti-PYCARD (ASC, 1:100, Abways Technology, Inc., AY0406, Shanghai, China), rabbit anti-CD31 (1:100, affinity, P16284, Changzhou, China), rabbit anti-α-tubulin (1:1000, Abways, AB0048, Shanghai, China), rabbit anti-γ-H2AX (1:100, Proteintech, 10856-1-AP, Wuhan, China) were incubated for 5 h, respectively. After triple washed by PBS, NLRP3, and CD31 were labeled by the second fluorescent antibody Cy3–conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (1:100, Proteintech, SA00009-2, Wuhan, China), and ASC, α-tubulin, γ-H2AX were labeled by the second fluorescent antibody Goat Anti-Rabbit IgG (H + L) Alexa Fluor 488 (1:100, Abways Technology, Inc., AB0141, Shanghai, China) for 2 h, respectively.
4
Immunohistochemical Analysis of Mouse Brain
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Mouse brain sections were incubated in 0.1% Triton (BS084, Biosharp, Beijing, China) in phosphate buffer solution for 30 min. After being rinsed, 3% BSA (BS114) was utilized to avoid non-specific reaction on mouse sections at RT for 30 min. A pre-dilution primary antibody (1:50–1:150) was dropped onto the brain sections and kept at 4°C overnight. Next day, all the brain sections were washed with PBS and incubated with second antibody (peroxidase-labbed IgG) at 37°C water bath for 1 h. A mixture of 0.05% 3,30-diaminobenzidine (DAB) and 0.01% H2O2 was dropped onto the brain sections for color reactions. At least 5 fields of the section were selected and analyzed for quantitative assessment.
5
Immunofluorescence Imaging of Lipid Droplets
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Marc-145 cells treated with different conditions were collected and immediately treated with 4% paraformaldehyde (BL539A, Biosharp) for 20 min. Cells were permeabilized in 0.1% Triton X-100 (BS084, Biosharp) for 15 min and then incubated with 5% bovine serum albumin for 1 h. After blocking nonspecific binding, cells were incubated with specific antibodies overnight at 4°C. The cells were washed and incubated with secondary antibodies for 1 h. The LDs were stained with BODIPY493/503 for 10 min, the nuclei were stained with DAPI (BL105A, Biosharp) for 10 min. Finally, all samples were analyzed using the Leica TCS SP8 fluorescence microscope (Leica, Germany).
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