Pancreas was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned by Histology Core Facility at Cornell and University of Michigan. For Hematoxylin/eosin staining, ten-micrometer-thick paraffin sections were stained and imaged using Aperio Scanscope. For immunostaining, paraffin-embedded sections were rehydrated with sequential wash in xylene, 100%, 95%, 75% ethanol and water and boiled in 1 mM EDTA for antigen retrieval. Subsequently, sections were blocked using 5% donkey serum and incubated at 4 °C overnight with primary antibodies: Anti-insulin (Abcam ab7842 or Linco 4011, 1:200), glucagon (Sigma K79bB10, 1:1000), Ki-67 (Abcam ab15580, 1:50), Pcna (Santa Cruz sc-56, 1:100), CD31 (Santa Cruz sc-1506, 1:100), Pdx1 (Cell Signaling D59H3, 1:100), Cdk4 (Santa Cruz sc-260, 1:100) and Ccnd2 (Santa Cruz sc-593, 1:100). Following day, slides were washed and incubated with conjugated secondary antibodies and mounted on slides with prolong gold antifade/ DAPI for nuclear staining. Fluorescence Images were captured under a Nikon A1 confocal microscope at Brehm Diabetes Research Center Imaging Facility at University of Michigan. For horseradish peroxidase enzyme (HRP) staining, slides were stained with Histostain kit and DAB substrate from Invitrogen.
Sc 260
Manufactured by Santa Cruz Biotechnology
The Sc-260 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is a compact and versatile device designed for various applications in the field of biotechnology and life sciences research. The Sc-260 is capable of performing essential tasks, such as sample preparation, analysis, and processing. The core function of the Sc-260 is to provide researchers with a reliable and efficient tool to support their experimental workflows. For more detailed information, please consult the product specifications or technical documentation.
Automatically generated - may contain errors
Lab products found in correlation
Dab substrate, by Thermo Fisher Scientific (2 mentions)
Histostain kit, by Thermo Fisher Scientific (2 mentions)
Ab7842, by Abcam (2 mentions)
Ab15580, by Santa Cruz Biotechnology (2 mentions)
A1 confocal microscope, by Nikon (2 mentions)
Linco 4011, by Merck Group (2 mentions)
K79bb10, by Abcam (2 mentions)
Sc 56, by Santa Cruz Biotechnology (2 mentions)
Sc 1506, by Cell Signaling Technology (2 mentions)
Sc 593, by Santa Cruz Biotechnology (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sc 7164, by Santa Cruz Biotechnology (1 mentions)
P21 sc 397, by Santa Cruz Biotechnology (1 mentions)
Gapdh sc 25778, by Santa Cruz Biotechnology (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Complete mini, by Roche (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Phosstop, by Roche (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
8 protocols using sc 260
1
Histological Analysis of Pancreatic Tissue
2
Comprehensive Molecular Analysis of HCC
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RT-qPCR of 13 HCC samples and related non-tumor counterparts were analyzed as described previously (17 (link)). The primers are exhibited in Table I . For other analyses, RNA was collected from cultured cells with the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions.
Nine HCC samples and the related non-tumor-counterparts were further used in western blot analysis as described previously (15 (link)). The following primary antibodies were used in western blot analysis: Rabbit polyclonal antibodies against Numb, cyclin-dependent protein kinase 4 (CDK4) (sc-260), S-phase kinase-associated protein 2 (SKP2) (sc-7164), Bcl-2 homologous antagonist/killer (BAK) (sc-832), cyclin-dependent kinase inhibitor 1 (p21) (sc-397) and GAPDH (sc-25778) (all from Santa Cruz Biotechnology, Inc.).
Thirty primary HCC tumors and their paired non-tumorous tissues were investigated by MSP, as described previously (18 (link)).
Nine HCC samples and the related non-tumor-counterparts were further used in western blot analysis as described previously (15 (link)). The following primary antibodies were used in western blot analysis: Rabbit polyclonal antibodies against Numb, cyclin-dependent protein kinase 4 (CDK4) (sc-260), S-phase kinase-associated protein 2 (SKP2) (sc-7164), Bcl-2 homologous antagonist/killer (BAK) (sc-832), cyclin-dependent kinase inhibitor 1 (p21) (sc-397) and GAPDH (sc-25778) (all from Santa Cruz Biotechnology, Inc.).
Thirty primary HCC tumors and their paired non-tumorous tissues were investigated by MSP, as described previously (18 (link)).
3
Histological Analysis of Pancreatic Tissue
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Pancreas was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned by Histology Core Facility at Cornell and University of Michigan. For Hematoxylin/eosin staining, ten-micrometer-thick paraffin sections were stained and imaged using Aperio Scanscope. For immunostaining, paraffin-embedded sections were rehydrated with sequential wash in xylene, 100%, 95%, 75% ethanol and water and boiled in 1 mM EDTA for antigen retrieval. Subsequently, sections were blocked using 5% donkey serum and incubated at 4 °C overnight with primary antibodies: Anti-insulin (Abcam ab7842 or Linco 4011, 1:200), glucagon (Sigma K79bB10, 1:1000), Ki-67 (Abcam ab15580, 1:50), Pcna (Santa Cruz sc-56, 1:100), CD31 (Santa Cruz sc-1506, 1:100), Pdx1 (Cell Signaling D59H3, 1:100), Cdk4 (Santa Cruz sc-260, 1:100) and Ccnd2 (Santa Cruz sc-593, 1:100). Following day, slides were washed and incubated with conjugated secondary antibodies and mounted on slides with prolong gold antifade/ DAPI for nuclear staining. Fluorescence Images were captured under a Nikon A1 confocal microscope at Brehm Diabetes Research Center Imaging Facility at University of Michigan. For horseradish peroxidase enzyme (HRP) staining, slides were stained with Histostain kit and DAB substrate from Invitrogen.
4
Quantitative Immunoblotting of Aortic Proteins
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Mouse aortic homogenates were suspended in lysis buffer (20 mM Tris pH 7.4, 2.5 mM EDTA, 1% Triton X-100, 1% deoxycholic acid, 0.1% sodium dodecyl sulfate, 100 mM NaCl, 10 mM NaF, 1 mM Na3VO4) containing protease and phosphatase inhibitors (Complete Mini, EDTA-free and PhosSTOP, Roche). These suspensions were then sonicated and clarified by centrifugation. Protein (20 or 40 μg) was loaded per lane and separated by SDS-PAGE. Immunoblotting was performed with antibodies directed against ACTA1 (Thermo Fisher RB-9010-P, rabbit polyclonal, 1:500), CALD1 (Sigma C4562, mouse monoclonal, 1:1000), CNN1 (Sigma C2687, mouse monoclonal, 1:5000), PPARγ (custom rabbit polyclonal raised against peptide CEKTQLYNRPHEEPSNS, 1:2000), and CDK4 (Santa Cruz sc-260, rabbit polyclonal, 1:500) as reported [25 (link)]. Relative levels of immunoreactive proteins were quantified using the ChemiDoc XRS imaging system and Quantity One software (Bio-Rad).
5
Protein Expression Analysis of Numb and Erk1/2
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Immunoprecipitated proteins and NCSC lysate were separated by SDS PAGE. For immunoblotting, antibodies used were as follows: anti-HA, anti-Numb, anti-Erk1/2 and anti-pErk1/2 (2756, 4695 and 4370, Cell Signaling Technology; 1:100, 1:1000, 1:1000, 1:1000, respectively), anti-β-actin (A5316, Sigma; 1:1000), anti-Numbl, anti-Lamin B1 and anti-GAPDH (10111, 12987 and 10494, Proteintech; 1:1000, 1:2000, 1:5000, respectively), and anti-Dll1 IC-termius anti-acetylated α tubulin, anti-Cdk2 and anti-Cdk4 (sc-9102, sc-23950, sc-163 and sc-260, Santa Cruz Biotechnology; 1:100, 1:500, 1:200, 1:200, respectively). Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Jackson ImmunoResearch. Each of the protein bands were visualized. For the detection of Numb, Erk1/2 and pErk1/2 protein, the same amount of each cell lysate protein was loaded. The Numb signals were calculated as fold change from D1ICD-expressing NCSCs compared with control NCSCs normalized with β-actin signal. The fold change of pErk1/2 was calculated from the phosphorylated Erk1/2 signals per Erk1/2 signal using same cell lysates.
6
Western Blot Analysis of Prostate Samples
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Protein extraction from frozen prostate samples for Western blot analysis has previously been described [49 (link)]. Western blots were prepared using 15–20 µg of protein lysate, blocked with 5% BSA in 1 × TBS / 0.1% Tween-20 for 1 h and incubated at 4 °C overnight with primary antibodies against AR (1:1000, ab133273, Abcam), p16INK4A (1:2000, ab211542, Abcam), CDK4 (1:200, sc-260, Santa Cruz), p53 (1:1000, CST#2524, Cell Signaling), β-Actin (1:1000, CST#4967, Cell Signaling) and β-Tubulin (1:2000, CST#2146, Cell Signaling). Western blots were quantified using ImageJ2.
7
Profiling Myc Phosphorylation in Tissues
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For IP-Western blots, 350μg of protein from lysates was used, and MYC was pulled down with the N262 antibody (sc-764; 2μg/sample). Western blot analysis was performed as described previously [21 (link)]. Immunoblots were visualized using the Odyssey IR imager (LI-COR) that can detect both Fluor 680 and IRDye 800 secondary antibodies (1:10000). Additional antibodies used for western blot include: monoclonal pS62-MYC (1:500, Abcam), Total MYC (Y69) (1:1000, ab32072, Abcam), B56α (1:500, ab72028, Abcam), β-Actin (1:10000, A5441, Sigma) and GAPDH (1:10000, AM4300, Ambion).
Mouse tissues were collected and fixed in 10% formalin-neutral buffer. Paraffin embedding and Hematoxylin and Eosin (H&E) staining were performed at OHSU Histopathology Core. Antibodies used for IF or IHC: pS62-MYC antibody (1:100) we developed as previously described [11 (link)] and pS62-MYC (1:500, ab185656, Abcam), Ki67 antibody (1:1000, Novocastra, NCL-Ki67-MM1), anti-BrdU (1:200, MCA2060, AbD serotec), Cdk4 (1:50, sc-260, Santa Cruz Biotechnology), and CD3 (1:100, Dako). IHC images were scanned and quantified by Aperio ImageScope 11.2.0.780 (Aperio Technologies). Antibodies used for the Flow Cytometry assay was described previously [35 (link)].
Mouse tissues were collected and fixed in 10% formalin-neutral buffer. Paraffin embedding and Hematoxylin and Eosin (H&E) staining were performed at OHSU Histopathology Core. Antibodies used for IF or IHC: pS62-MYC antibody (1:100) we developed as previously described [11 (link)] and pS62-MYC (1:500, ab185656, Abcam), Ki67 antibody (1:1000, Novocastra, NCL-Ki67-MM1), anti-BrdU (1:200, MCA2060, AbD serotec), Cdk4 (1:50, sc-260, Santa Cruz Biotechnology), and CD3 (1:100, Dako). IHC images were scanned and quantified by Aperio ImageScope 11.2.0.780 (Aperio Technologies). Antibodies used for the Flow Cytometry assay was described previously [35 (link)].
8
Western Blot Analysis of Cell Signaling
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Whole-cell lysates were prepared as previously described31 (link). Protein detection was performed using primary antibodies detecting p53 (DO1, Santa Cruz, 1:1,000), p21 (Sc-397, Santa Cruz, 1:1,000), CDK4 (Sc-260, Santa Cruz, 1:1,000), phospho-histone H3 (ser 10) (9701, Cell Signaling, 1:100). Proteins were visualized using adequate secondary antibody (Dako) and ECL reagents (GE Healthcare).
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