Panc-02 and Panc-02-PTX cells were fixed with 4% paraformaldehyde after treatment or transfection at the indicated time points. Then we permeabilized the cells with 0.1% Triton100. After that cells were immersed three times in Tris-buffered saline (TBS) and incubated with the PHB1 primary antibody (sc-28259, diluted 1:200; Santa Cruz Biotechnology, TX) overnight at 4°C and the corresponding Cy3 goat anti-rabbit secondary antibodies (3101, diluted 1:150; Earthox, Millbrae, CA, USA) for 1 h at room temperature (RT). DAPI (diluted 1:1,000; Sigma-Aldrich) was used to stain the nucleus. Tissue sections were performed on a microtome (HM 430, MICROM International, Walldorf, Germany). Sections were blocked with 5% BSA in PBS for 30 min and incubated with the primary mouse monoclonal antibody anti-FoxM1 (sc-271746, diluted 1:100; Santa Cruz Biotechnology) or polyclonal rabbit antibody against PHB1 (sc-28259, diluted 1:200; Santa Cruz Biotechnology), respectively, at 4°C overnight. The primary antibodies were detected with the secondary antibodies (sheep anti-mouse or goat anti-rabbit). After immunolabeling, the tissue samples were incubated with 1 μg/mL DAPI and covered in 0.1% 1, 4-diazabicyclo [2.2.2] octane (DABCO; Sigma-Aldrich). Microscopic images of cells were obtained using an Olympus IX71.
Sc 271746
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-271746 is a laboratory product offered by Santa Cruz Biotechnology. It serves as a general-purpose tool for researchers and scientists. The core function of Sc-271746 is to facilitate various experimental procedures within the context of life science research and analysis. Detailed specifications and intended use are not provided in order to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
1 4 diazabicyclo 2.2.2 octane dabco, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Sc 28259, by Santa Cruz Biotechnology (1 mentions)
Ab180710, by Abcam (1 mentions)
Ab9566, by Abcam (1 mentions)
Secondary abs, by Thermo Fisher Scientific (1 mentions)
Tcs tiv, by Leica (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Bioruptor next gen sonicator, by Diagenode (1 mentions)
4 protocols using sc 271746
1
Immunofluorescence Analysis of PHB1 and FoxM1 Expression
2
Quantifying FoxM1 and β-Catenin in Kidney Cells
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Double immunofluorescence staining was implemented as established protocols.10 The mouse kidney sections were incubated with anti‐FoxM1 (ab1807.10; Abcam) and anti‐AQP1 (ab9566; Abcam). NRK‐52E cells were incubated with adenovirus expressing rat FoxM1 gene (Ad‐FoxM1) or empty vector (Ad‐null), then followed by fixation at 37°C using 4% paraformaldehyde for 20 minutes. Fixed cells were hybridized with primary antibodies against FoxM1 (sc‐271746; Santa Cruz) and active β‐catenin (05‐665; Millipore) at 4°C overnight. Then, secondary antibodies (Proteintech) and 4′,6‐Diamidino‐2‐phenylindole (DAPI; Beyotime) visualized primary antibodies and nuclei, respectively. Cells were viewed using a confocal biological microscope (ZEISS).
3
Protein Expression Analysis by Immunofluorescence
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The sections were immobilized using 4% paraformaldehyde and permeabilized for 20 minutes with 0.5% Triton X-100. Next, the sections were blocked with 5% BSA for 1 hour at room temperature and incubated with primary anti-FOXM1 (1:50; catalog sc-271746, Santa Cruz) and anti-RNF112 (1:50; catalog A15333, Abclonal) overnight at 4°C. Finally, the sections were incubated with secondary Abs (1:300; Invitrogen) for 1 hour at 37°C. Cell nuclei were stained with DAPI (Beyotime). All images were collected by confocal laser microscopy (TCS-TIV, Leica).
4
FOXM1 Chromatin Immunoprecipitation Protocol
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To cross‐link proteins to DNA, H1299 and PC9 cells were treated for 10 min at 37 °C with 0.7% formaldehyde. Cross‐linking was quenched by adding glycine to the culture medium at a final concentration of (0.125 M) for 5 min. The cells were sonicated using a Bioruptor Next Gen sonicator (Diagenode, Denville, NJ, USA) for 5 min in 30 s on/30 s off cycles to obtain ≈500 bp DNA fragments. Sonicated cell lysates containing genomic DNA fragments were precleared to eliminate unwanted non‐specific components. ChIP was performed using antibodies against FOXM1 (#sc‐271746, Santa Cruz Biotechnology) with rotation at 4 °C overnight, followed by various washing steps, decrosslinking, and digestion of proteins with proteinase K (#P2308; Sigma‐Aldrich, St. Louis, MO, USA). The immunoprecipitated DNA fragments were purified using the phenol–chloroform method. The purified DNA pellets were resuspended in TE buffer (pH 8.0) and used for PCR. Primers specifically detecting the FOXM1 binding region were designed using the Primer3 tool for ChIP‐PCR (Table S4 , Supporting Information).
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