The brain slices were washed 3 times with PBS for 5 min each time and then treated with 0.1% Triton X-100 (Beyotime Biotechnology, ST795, Shanghai, China) diluted with PBS for 20 min. Then, slices were placed in 5% BSA (Beyotime Biotechnology, ST023, Shanghai, China) diluted with PBS and sealed at room temperature for 1 h. After incubating overnight with the primary antibody [rabbit antibody PSD95 (Abcam, ab238135, Cambridge, UK, 1:200), rabbit antibody IBA1 (Fujifilm, 019-19741, Tokyo, Japan, 1:300)] at 4 °C, the sections were washed 3 times with PBS. A secondary antibody [goat anti-rabbit IgG H&L (Alexa Fluor® 488) (Abcam, ab150077, 1:500)] was then applied at room temperature for 1 h, and the slices were washed in PBS 3 times. Finally, the nuclei were restained with 1 μg/mL DAPI (Beyotime Biotechnology, C1002, Shanghai, China) for 10 min, and the slides were mounted with an anti-fluorescence quenching seal. The slides were viewed with a fluorescence microscope (BioTek, Cytation5, Hong Kong, China) and all images were collected using a microscopic imaging system (BioTek, Cytation5, Hong Kong, China). The ImageJ (v1.54f) application was used to analyze the collected images.
St023
Manufactured by Beyotime
Sourced in China
The ST023 is a compact and versatile laboratory centrifuge designed for a range of applications. It features a maximum speed of 6,000 rpm and a maximum relative centrifugal force (RCF) of 2,683 g. The centrifuge accommodates fixed-angle rotors with a capacity of up to 6 x 15 mL tubes. Its user-friendly interface and safety features make it a reliable instrument for various laboratory tasks.
Automatically generated - may contain errors
Lab products found in correlation
Np 40 lysis buffer, by Beyotime (2 mentions)
Ab238135, by Abcam (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Cytation 5, by Agilent Technologies (1 mentions)
St795, by Beyotime (1 mentions)
Ab150077, by Abcam (1 mentions)
C1002, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
T8200, by Solarbio (1 mentions)
Horseradish peroxidase conjugated anti mouse rabbit igg, by ZSGB-BIO (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Gapdh, by Beyotime (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
St506, by Beyotime (1 mentions)
5 protocols using st023
1
Immunohistochemical Analysis of Brain Slices
2
Preparation of COP-22-loaded BSA Nanoparticles
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Previous reported similar method was utilized to prepare 22 NPs [41 (link)]. First, 10 mg of COP-22 and 50 mg of the egg yolk lecithin E80 (E80) (Yuanye, S30871, Shanghai, China) in 2 mL of methanol were stirred for 1 h at 40 °C to gain the clear solution. After stirring, methanol was removed using the rotary evaporator, and then, COP-22/E80 complexing agent was formed. This COP-22/E80 complexing agent was re-dissolved in 1 mL of methylene chloride, as the organic phase. Next, 200 mg of BSA (Beyotime, ST023, Shanghai, China) was dissolved in 10 mL ddH2O, as the aqueous phase. Additionally, the organic phase was slowly added dropwise to the aqueous phase under ultrasonic waves, and the mixture was sonicated for another five minutes. Finally, the COP-22-loaded BSA nanoparticles (22 NPs) were constructed after the removal of organic solvent.
3
Immunofluorescence Staining of Cultured Cells
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Cells grown on glass coverslips in a 24-well plate were fixed in 4% paraformaldehyde (Keshi, 30525-89-4) for 20 min, permeabilized with 0.2% Triton X-100 (in PBS, Solarbio, T8200) for 20 min, and then blocked with 5% BSA (in PBS, Beyotime, ST023) for 2 h at room temperature (RT). Primary antibodies were incubated with cells overnight at 4°C and then with Alexa Fluor-conjugated secondary antibodies for 1 h at 37°C. Next, the cells were counterstained with DAPI (4′,6′-diamidino-2-phenylindole, 1:100; Solarbio, C0060) in the dark for 10 min to visualize the nuclei. Finally, the tablets were sealed using an anti-fluorescence quenching agent (Beyotime, P0128S).
Images were captured with a confocal laser scanning microscope at 400× magnification (eyepiece 10×, objective 40×). Photo software: OlyVIA; the fluorescence microscope (80i) and microscope lens (including color filter, E400) were from Nikon, Japan. The software used for localization statistics was ImageJ.
Images were captured with a confocal laser scanning microscope at 400× magnification (eyepiece 10×, objective 40×). Photo software: OlyVIA; the fluorescence microscope (80i) and microscope lens (including color filter, E400) were from Nikon, Japan. The software used for localization statistics was ImageJ.
4
Western Blot Analysis of Cellular Proteins
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Total cellular protein was extracted using the NP‐40 lysis buffer (P0013F; Beyotime Biosciences, Shanghai, China) containing a protease‐inhibitor PMSF (ST506; Beyotime Biosciences, Shanghai, China) and quantified using an enhanced BCA Protein Assay Kit (P0010; Beyotime Biosciences, Shanghai, China). Proteins (30 μg/lane) were resolved by 10% SDS‐PAGE gel, and transferred to 0.45 or 0.22 μm PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% BSA (st023; Beyotime Biosciences, Shanghai, China), and then incubated overnight at 4°C with the appropriate primary antibodies: anti‐TMEM107 (1:1000; Abcam; ab181396), E‐cadherin (1:500; cell signaling technology; 3195), N‐cadherin (1:500; Cell Signaling Technology; 13 116), vimentin (1:500; Cell Signaling Technology; 5741), MMP2 (1:500; Cell Signaling Technology; 87809), MMP9 (1:500; Cell Signaling Technology; 13667), and GAPDH (1:1000; Beyotime; AF0006). Membranes were washed with Tris‐buffered saline with Tween‐20 and then incubated with horseradish peroxidase‐conjugated anti‐mouse/rabbit IgG (1:2000; ZSGB‐BIO, Beijing, China) at room temperature for one hour. Target proteins were detected by ECL (Thermo Fisher Scientific, Waltham, MA, USA) using a BioImaging system (UVP, Inc., Upland, CA, USA).
5
Western Blot Analysis of ASC Protein Expression
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Cultured ASCs were lysed on ice in NP-40 Lysis Buffer (P0013F, Beyotime) containing protease and phosphatase inhibitor. Lysate protein concentrations were determined with Micro BCA Protein Assay Kit (23235, Thermo Scientific). In total, 200–400 μg of protein per sample (n = 3) was used for western blotting analysis. Proteins were separated by 4%–12% Bis-Tris Bolt gels (NW04120BOX, Invitrogen) and transferred to pure nitrocellulose blotting membranes (66485, Pall Life Sciences). Membranes were blocked with 5% nonfat milk (P0216, Beyotime) or 3% bovine serum albumin (ST023, Beyotime) in Tris-Buffered Saline with Tween-20 (ST671, Beyotime) at room temperature for 1 h. Target proteins were incubated overnight at 4 °C with the following primary antibodies: CLEC3B (ab51883, Abcam), Type I Collagen (ab138492, Abcam), SMAD-2/3 (3102S, Cell Signaling Technology), and phosphorylated SMAD-2/3 (8828S, Cell Signaling Technology). HRP-conjugated corresponding secondary antibodies (ZB-2301/2305, ZSGB-BIO) were used at room temperature for 1 h, followed by chemiluminescent detection (P0018AM, Beyotime). Equal loading controls were indicated with GAPDH (2118L, Cell Signaling Technology). Densitometry analysis was performed by quantifying the intensity of the bands using the ImageJ software.
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