Briefly, the human NP tissues embedded in paraffin were cut in 15 μm sections. Then, the sections were deparaffinized with environment-friendly de-paraffin liquid (G1128, Servicebio, China) and dehydrated using gradient alcohol. The membrane-breaking solution (G1204, Servicebio, China) was used under the protocols. The sections were subsequently incubated with 3% BSA for 25 min to block the endogenous peroxidase, then with primary antibody against Sirt3 (#AF5135, Affinity, China, 1:200), GPX4 (#DF6701, Affinity, China, 1:200), FTH (#DF6278, Affinity, China, 1:50), ADAMTS5 (#DF13268, Affinity, China, 1:150), and MMP3 (#AF0217, Affinity, China, 1:100), at 4 °C overnight. On the second day, the sections were incubated with HRP-conjugated Goat Anti-Rabbit IgG H&L (511,203, ZENBIO, China, 1:300) for 1 h, finally the counterstaining was performed with hematoxylin solution for 5 min. The images of stained sections were obtained using the light microscopy (BX43, Olympus, Japan).
Af0217
Manufactured by Affinity Biosciences
Sourced in China
AF0217 is a piece of laboratory equipment used for cell culture applications. It is designed to provide a controlled environment for the growth and maintenance of cells.
Automatically generated - may contain errors
Lab products found in correlation
Df6278, by Affinity Biosciences (1 mentions)
Af5135, by Affinity Biosciences (1 mentions)
G1128, by Wuhan Servicebio Technology (1 mentions)
Df13268, by Affinity Biosciences (1 mentions)
Hrp conjugated goat anti rabbit igg h l, by ZenBio (1 mentions)
G1204, by Wuhan Servicebio Technology (1 mentions)
Df6701, by Affinity Biosciences (1 mentions)
Af0135, by Affinity Biosciences (1 mentions)
G1401, by Wuhan Servicebio Technology (1 mentions)
Af0198, by Affinity Biosciences (1 mentions)
Df7561, by Affinity Biosciences (1 mentions)
Af5006, by Affinity Biosciences (1 mentions)
Ds ri2, by Nikon (1 mentions)
Af1027, by Affinity Biosciences (1 mentions)
2 protocols using af0217
1
Immunohistochemical analysis of protein expression
2
Immunofluorescent Analysis of HNP Cell Markers
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HNP cells were immunofluorescently stained for aggrecan (#DF7561, Affinity, China, 1:200), type II collagen (#AF0135, Affinity, China, 1:200), MMP3 (#AF0217, Affinity, China, 1:200), Ki-67 (#AF0198, Affinity, China, 1:200), p65 (#AF5006, Affinity, China, 1:200), and TGF-β1 (#AF1027, Affinity, China, 1:200), following a previously described protocol28 (link). Briefly, samples were fixed with 4% PFA for 25 minutes and permeabilized with 0.1% vol/vol Triton X-100 for 10 minutes. Samples were then blocked with 5% BSA at room temperature for 60 minutes. Following this, the samples were incubated with the appropriate primary antibodies at 4°C overnight. The HNP cells were washed three times with cold PBS and incubated with a 1:500 dilution of a secondary antibody (550,076, Zen Bio, China) at room temperature for 60 minutes. Nuclei visualization was performed using DAPI. Finally, HNP cells were sealed with an anti-fluorescence quencher (G1401, Servicebio, China) and observed under a fluorescence microscope (DS-Ri2, Nikon, Japan).
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