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 56
Manufactured by Santa Cruz Biotechnology
Sourced in United States, China
Sc-56 is a laboratory equipment product offered by Santa Cruz Biotechnology. The core function of Sc-56 is to facilitate sample preparation and analysis in scientific research and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
A1 confocal microscope, by Nikon (3 mentions)
Dxm1200f, by Nikon (3 mentions)
Hematoxylin, by Vector Laboratories (3 mentions)
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)
Linco 4011, by Merck Group (2 mentions)
K79bb10, by Abcam (2 mentions)
Sc 1506, by Cell Signaling Technology (2 mentions)
Sc 260, by Santa Cruz Biotechnology (2 mentions)
Sc 593, by Santa Cruz Biotechnology (2 mentions)
Bond max, by Leica (2 mentions)
Ab15580, by Abcam (2 mentions)
Sc 23900, by Santa Cruz Biotechnology (2 mentions)
Axioplan microscope, by Nikon (2 mentions)
Ab1791, by Abcam (2 mentions)
Sc 18917, by Santa Cruz Biotechnology (2 mentions)
Nb100 304, by Novus Biologicals (2 mentions)
Cd206, by Abcam (2 mentions)
55 protocols using sc 56
1
Histological Analysis of Pancreatic Tissue
2
Immunohistochemical Evaluation of VEGF and PCNA
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The 4-μm sections were made and placed on positively charged slides for immunohistochemical (IHC) staining. Then, these slides were stained with the VEGF (VG-1, Code; SC-53462, Santa Cruz, dilution rate 1:50) and PCNA (PCNA, Code: SC-56, Santa Cruz, dilution rate 1:50) antibodies. A fully automated IHC device (Leica Bond-Max, Melbourne, Australia) was used for immunostaining. Histopathological and immunohistochemical evaluations were done using a light microscope (Olympus BX53, Tokyo, Japan). For VEGF antibody staining, pneumocytes and macrophages of human lung tissue were used as a positive control. For PCNA antibody, the staining of hepatocytes in human liver tumor was used as a positive control. Negative controls were tested by omission of the primary antibody (secondary only). The slides were then covered with coverslips and treated with alcohol and xylene. VEGF and PCNA immunostaining ratios were defined in the range of 0 to 3: Score 0=no staining, Score 1=weak staining (less than 10.0% focal involvement), Score 2=moderate staining (11.0–50.0% regional involvement), and Score 3=strong staining (greater than 50.0% diffuse involvement) [20 (link)].
3
Quantifying Osteoclast Differentiation
4
Quantification of PCNA Immunolabeling
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Paraffin sections of 3-μm thickness were stained with antibodies to α smooth muscle actin (α-SMA) (dilution 1:900, clone 1A4, Sigma, St. Louis, MO, USA), von Willebrand factor (vWF) (dilution 1:900, Dako, Hamburg, Germany) and proliferating cell nuclear antigen (PCNA) (dilution 1:200, sc-56, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Automated quantification of PCNA immunopositive labeling was performed using QuPath [29 (link)]. The software was trained to recognize PCNA-stained nuclei and positive-labeled cells using positive cell and subcellular detection modules. The number of cells with PCNA-positively labeling per μm2 and the percentage of cells detected with PCNA immunolabeling were recorded and compared between all groups.
5
Chromatin Immunoprecipitation and Western Blot Analysis
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Indirect IF, WB and ChIP assays were carried out according to standard protocols. Antibodies used were a rabbit polyclonal anti-TRF2 (Novus Biologicals, NB110-57130, IF dilution 1:500, WB 1:2,000), a rabbit polyclonal anti-TRF1 (kind gift from J. Karlseder, WB 1:1,000), a rabbit polyclonal crude serum anti-TRF1 (kind gift from J. Lingner, ChIP 1:500), the mouse monoclonal S9.6 (kind gift from S. Leppla, IF 1:1,000), rabbit polyclonals anti-RNAseH1 and anti-Lamin B1 (GeneTex GTX117624, WB 1:500; GTX103292S, WB 1:1,000), rabbit polyclonals against total RPA32, pSer33, total KAP1, phosphorylated serine S2 or serine S5 from human RNAPII C-terminal domain (Bethyl Laboratories A300-244A, WB 1:5,000; A300-246A WB and IF 1:1,000; A300-274A, WB 1:5,000; A300–654A, ChIP 1:500; A300–655A, ChIP 1:500), mouse monoclonals anti-PCNA and PML (Santa Cruz Biotechnology sc-56, WB 1:5,000; sc-966, IF 1:250).
6
Immunohistochemical Profiling of Tissue Markers
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Rabbit anti-Tissue Factor antibody (1:200, ab228968; Abcam), rabbit anti-AQP5 antibody (1:200, ab92320; Abcam), rabbit anti-Ki67 (1:200, ab15580; Abcam), mouse anti-PCNA (1: 300, sc-56; Santa Cruz), mouse anti-HK-Atpase-β (1:300, sc-374094; Santa Cruz), rabbit anti-E-cadherin (1: 300, 3195; Cell Signaling), rabbit anti-CD74 (1:300, ab64772; Abcam) and mouse anti-E cadherin (1:200, 610182; BD Biosciences). FITC-conjugated UEAI lectin (1:2000, L32476; Thermo Fisher) and Lectin GS-II conjugate with Alexa Fluor 647 (100 μg/mL, L32451; Thermo Fisher).
7
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.
8
Immunohistochemical Analysis of Liver Tissue
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Livers were fixed and paraffin embedded. Sections were routinely stained with Hematoxylin&Eosin (7-μm) or incubated with mAb anti-PCNA antibody (PC10) (1:200 dilution, sc-56, Santa Cruz Biotechnology) as previously indicated [53 (link)]. The slices were examined with a Zeiss Axioplan microscope equipped with a Nikon DXM1200F digital camera. The PCNA cell count was quantified in four randomly selected fields from each animal and analyzed using ImageJ software. Ki-67 staining was performed using a specific antibody (sc-23900, 1:200 mouse) from Santa Cruz Biotechnology.
9
Cell Lysis and Western Blot Analysis
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The standard protocol (described above) for cell lysis and western blot was followed with exception that the membrane washes were in tris-buffered saline with 0.1% Tween 20 (TBS-T) and the membrane blocking and antibody probing was performed with 5% BSA in TBS-T. The primary antibodies used for protein detection were rabbit monoclonal anti-Aurora A (1:10,000, ab52973, Abcam), mouse monoclonal anti-PCNA (1:4,000, PC10, sc-56, Santa Cruz Biotechnology), rabbit monoclonal anti-phospho-histone H3 (pSer10) (1:10,000, 04-817, Merck) and rabbit polyclonal anti-histone H3 (1:10,000, ab1791, Abcam). The secondary antibodies were goat anti-rabbit and goat anti-mouse (31460 and 31430, Thermo Scientific) diluted 1:10,000 in TBS-T containing 5% BSA. The immunoblot quantification was performed by ImageJ software (NIH) and the data for normalization to the loading control were obtained following the integration of the band intensity. Unprocessed immunoblot scans for AURKA expression are provided in Supplementary Fig. 9 .
10
Immunohistochemical and Immunofluorescence Analyses of Tumor Samples
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Formalin-fixed and paraffin-embedded tumor samples were cut into 3.5-µm sections, deparaffinized, and antigen-retrieved using sodium citrate solution (0.01 M, PH: 6.0). Then, the sections were processed for antibody staining for immunohistochemistry (IHC) or immunofluorescence (IF). For IHC, slides were incubated with primary anti-PCNA (1:250, sc-56, Santa Cruz), anti-Foxp3 (1:100, 12,653, Cell Signaling Technology) or anti-CD8 Ab (1:200, ab203035, Abcam), and then secondary antibody HPR anti-mouse or rabbit IgG (Maxim). The slides were colored with 3, 3ʹ-Diaminobenzidine (DAB, Sigma-Aldrich) and counterstained by hematoxylin. For IF, anti-CD31 (1:250, sc-376,764, Santa Cruz), anti-CD206 (1:1000, ab64693, Abcam) and anti-F4/80 (1:250, sc-377,009, Santa Cruz) were used as primary Abs while anti-mouse IgG (H + L) (1:1000, 4408, Cell Signaling Technology) and anti-rabbit IgG (H + L) (1:1000, 4413, Cell Signaling Technology) were used as secondary Abs. The sections finally were mounted using DAPI-Fluoromount-G clear mounting agents (Southern Biotech, Birmingham, UK) and images were taken using fluorescence microscopy (Nikon, Japan). Positive cells were quantified using Image J software and expressed as the mean of the percentage of positive cells ± SD with 10 randomly selected fields per sample.
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