Adherent chondrocytes at 70% confluency were grown on glass coverslips in 6-well plates and starved for 12 h before treatment. After fixation with 4% paraformaldehyde for 20 min, chondrocytes were permeabilized with 0.5% Triton X-100 buffer (Beyotime, Jiangsu, China) for 5 min, and then blocked with 1% bovine serum albumin at 4 °C for 10 min. Chondrocytes were cultured with SP-D antibody (1:400 dilution) for 2 h followed by incubated with Cy3-labeled secondary antibody (1:100 dilution; Boster Biological Engineering, Wuhan, China) in the dark for 1 h. Nuclei were counter-stained with DAPI (KeyGEN Biotech, Nanjing, China) for 10 min. Slides were visualized by an Olympus microscope, and the IOD of SP-D was calculated by Image-Pro Plus 6.0 image analysis software.
Triton x 100 buffer
Manufactured by Beyotime
Sourced in China
0.5% Triton X-100 buffer is a laboratory solution that contains 0.5% Triton X-100, a non-ionic detergent, in an aqueous buffer. It is commonly used in various biochemical and cell biology applications as a mild detergent to solubilize proteins and disrupt cell membranes.
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Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Keygen Biotech (1 mentions)
Anti claudin 1 antibody, by ABclonal (1 mentions)
Poly l lysine (pll), by Biosharp (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
2 protocols using triton x 100 buffer
1
Immunofluorescence Analysis of SP-D in Chondrocytes
2
Claudin-1 Distribution in IPEC-J2 Cells
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The distribution of the tight-junction protein (claudin-1) in the IPEC-J2 cells was determined by an immunofluorescence analysis. Briefly, the IPEC–J2 cells were seeded on cover-slides treated with poly(L-lysine) (Biosharp, Beijing, China) and placed in 12-well plates for 12 h to reach 70% confluence. The cells were fixed with 4% paraformaldehyde (Beyotime, Shanghai, China) for 30 min, and then they were permeabilized with 0.5% Triton X-100 buffer (Beyotime, Shanghai, China) at room temperature for 20 min. Thereafter, the IPEC–J2 cells were incubated with anti-claudin-1 antibody (dilution 1:500; ABclonal, Wuhan, China) for 1 h at room temperature and then incubated with fluorescein conjugated goat anti-rabbit IgG (H + L) antibody (dilution 1:500; Proteintech, Wuhan, China) in the dark for 1 h. The cell nuclei were stained with 4′,6-Diamidino-2-Phenylindole (DAPI, Beyotime, Shanghai, China) solution. The slides were visualized under a laser scanning confocal microscope (Zeiss, LSM 700; Oberkochen, Germany).
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