Lung cancer tissues and paracancerous tissues were embedded by paraffin and sectioned into 4‐μm‐thick sections. Paraffin‐embedded tissue sections were deparaffinized and hydrated. Citric acid (pH 6) was used to extract antigens for 30 minutes at 97°C after which the antigens were treated with 3% H2O2. Then, the tissue sections were incubated with the anti‐rabbit antibodies against FXYD3 (ab205534, 1:400, Abcam), KDM3A (ab191387, 1:500, Abcam) and DCLK1 (ab109029, 1:500, Abcam) overnight at 4℃. Afterwards, the sections were incubated with the horseradish peroxidase (HRP)‐conjugated secondary antibody (Dako; Agilent Technologies, Inc) for 30 minutes. Following 10‐minute incubation with 3,3'‐diaminobenzidine tetrahydrochloride, the sections were re‐stained with haematoxylin for 2 minutes. Finally, the pathology results were obtained under a microscope.
Ab205534
Manufactured by Abcam
Sourced in United States
Ab205534 is a primary antibody for detecting a specific protein target. It is designed for use in common immunoassay techniques. The product details and specifications are available on the Abcam website.
Automatically generated - may contain errors
Lab products found in correlation
Ab191387, by Abcam (1 mentions)
Ab109029, by Abcam (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
Ab76003, by Abcam (1 mentions)
Ab92536, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab32124, by Abcam (1 mentions)
Ab70892, by Abcam (1 mentions)
Ab97051, by Abcam (1 mentions)
Ab205719, by Abcam (1 mentions)
R0010, by Solarbio (1 mentions)
Ab81283, by Santa Cruz Biotechnology (1 mentions)
Image quant las 4000c, by GE Healthcare (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
4 protocols using ab205534
1
Immunohistochemical Analysis of Lung Cancer Markers
2
Protein Expression Profiling of Cancer Cells
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HeLa and CaSki cells were reaped and rinsed in PBS, followed by lysing in 100 µl of lysis buffer with protease inhibitor cocktail for 20 min on ice. The lysates were separated on the SDS-polyacrylamide gel and transferred into the PVDF membranes. Sealed with 5% defatted milk, samples were immunoblotted with the primary antibodies. The primary antibodies used were as below: anti-Bcl-2 (1:1000; ab32124, Abcam, Cambridge, MA, U.S.A.), anti-Bax (1:1000; ab32503, Abcam), anti-MMP2 (1:1000; ab92536, Abcam), anti-MMP9 (1:1000; ab76003, Abcam), anti-FXYD3 (1:1000; ab205534, Abcam), GAPDH (1:5000; ab9485, Abcam). Later, the samples were incubated with horseradish peroxidase-labeled secondary antibodies (1:5000; ab205718, Abcam), and the protein bands were directly viewed with an ECL detection reagent (Hercules, CA, U.S.A.), followed by analysis via ImageJ software.
3
Immunoprecipitation of Na+/K+ ATPase
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After solubilization in RIPA lysis buffer, protein extracts (0.5–1 mg) from cells were precleared and incubated overnight at 4°C with anti-α1 subunit Na+-K+ ATPase (D4Y7E; Cell Signaling Technology, USA), anti-FXYD3 (ab205534; Abcam, UK), or anti-β1 subunit (D6U8Q; Cell Signaling Technology, USA) Na+/K+-ATPase antibodies followed by precipitation for 2 h at 4°C with protein A/G plus agarose-coated beads (Abcam, UK). Sample buffer was added, the mixture was boiled for 5 min and sedimented, and the supernatant was used for immunoblotting.
4
Western Blot Analysis of Protein Expression
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Cells were lysed by radio immunoprecipitation assay (RIPA) lysis (R0010, Solarbio, Beijing, China), and the lysates were quantitated by BCA protein assay kit (GBCbio, Guangzhou, China). 40 µg protein sample was separated using sodium dodecyl sulphate polyacrylamide gel electrophoresis that had been electro‐transferred onto polyvinylidene fluoride (PVDF) membranes and probed with primary antibodies overnight at 4°C. Immunoblots were visualized with goat anti‐rabbit Immunoglobulin G (IgG; 1:2000, ab97051, Abcam) or goat anti‐mouse IgG (ab205719, 1:2000, Abcam) and enhanced chemiluminescence detection reagents and were captured under the Image Quant LAS 4000C (GE, USA General Electric Company, Schenectady, NY, USA) microscope. Primary antibodies used: KDM5A (Abcam, ab70892, 1:1000), FXYD3 (Abcam, ab205534, 1:1000), p‐p85 (Abcam, ab182651, 1:1000), p85 (CST, #4257 1:1000), p‐AKT (Abcam, ab81283, 1:1000), AKT (Santa Cruz, CA, USA, sc‐5298, 1:1000) and β‐Actin (Santa Cruz, sc‐8432, 1:1000).
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