Tissues were embedded in paraffin. Slices (5 μm) were treated in H2O2 (3%) dissolved in methanol and 5% normal horse serum before staining. Sections were incubated with the following primary antibodies overnight at 4 °C: anti-TRIM27 antibody (ab137638; Abcam, UK) and anti-NF-KB antibody (ab16502; Abcam, UK). The sections were then incubated with secondary goat anti-rabbit IgG (1:100; ab64256; Abcam) for 30 min at room temperature. The sections were visualized with diaminobenzidine and hematoxylin. Cells with > 25% positive staining were defined as having “high expression.”
Ab64256
Manufactured by Abcam
Sourced in United Kingdom
Ab64256 is a recombinant monoclonal antibody that recognizes the CD31 antigen. CD31, also known as PECAM-1, is a cell adhesion molecule expressed on the surface of endothelial cells and some leukocytes.
Automatically generated - may contain errors
Lab products found in correlation
Ab15580, by Abcam (4 mentions)
Ab137638, by Abcam (2 mentions)
Ab16502, by Abcam (2 mentions)
Ab64269, by Abcam (2 mentions)
Ab64238, by Abcam (2 mentions)
3 3 diaminobenzidine dab kit, by Vector Laboratories (2 mentions)
Anti p65 antibody sc 372, by Santa Cruz Biotechnology (2 mentions)
Ab9535, by Abcam (2 mentions)
Ab124760, by Abcam (2 mentions)
Peroxidase labeled anti rabbit antibody nif 824, by GE Healthcare (2 mentions)
Peroxidase labeled anti mouse antibody nif 825, by GE Healthcare (2 mentions)
Tcs sp5, by Leica (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
3 3 diaminobenzidine substrate, by Thermo Fisher Scientific (1 mentions)
Neutral buffered formalin, by Thermo Fisher Scientific (1 mentions)
Ab112, by Abcam (1 mentions)
Sh nc, by RiboBio (1 mentions)
Ab86607, by Abcam (1 mentions)
Ab76003, by Abcam (1 mentions)
Balb c nude mice, by Charles River Laboratories (1 mentions)
17 protocols using ab64256
1
TRIM27 and NF-kB Expression Analysis
2
PCNA expression analysis in tumor tissues
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Tumor tissues were fixed with 4% paraformaldehyde (Solarbio) and encapsulated in paraffin. IHC analysis was used to analyze PCNA expression as reported [30 (link)]. The sections (4 µm) of the tumors were hatched with diluted anti-PCNA antibody (ab29, 1:1000, Abcam) and biotinylated IgG secondary antibody (ab64256, 1:500 dilution, Abcam), stained with 3,3’-diaminobenzidine substrate (Invitrogen), and observed under microscopy (Leica TCS SP5) and photographed.
3
Quantifying Neuroanatomical Changes in Rodents
4
Inhibition of Tumor Growth by Targeting circ_0011946
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BALB/c nude mice (male, 5-week-old) were procured from Beijing Laboratory Animal Center (Beijing, China) and fed in specific pathogen-free microisolator cages. Lentiviruses expressing shRNA-circ_0011946 (sh-circ_0011946) and the negative control (sh-NC) were supplied by Ribobio (Guangzhou, China). Stable CAL27 cells were established by transducing with sh-circ_0011946 or sh-NC, and the selection of puromycin. Stable CAL27 cells (3 × 106/mouse) were hypodermically inoculated into the light flanks of the mice (n = 6/group). Tumor volume was estimated every week based on the formula: length × width2/2. 4 weeks later, all mice were euthanized using 5% isoflurane. Tumor tissues were collected and weighed. circ_0011946, miR-216a-5p and BCL2L2 levels in tumor samples were measured. Proliferation of the tumors was assessed with paraffin-embedded tumor tissues by immunohistochemistry under the standard method using the anti-Ki67 antibody (ab15580, 1:100 dilution, Abcam), biotinylated goat anti-rabbit IgG secondary antibody (ab64256, 1:300 dilution, Abcam) and a 3,3-diaminobenzidine (DAB) Kit (Vector Laboratories, Peterborough, UK). The animal experiments got the approval of the Institutional Animal Care and Use Committee of Jiaxing Hospital of Traditional Chinese Medicine, Jiaxing University.
5
Xenograft Formation and Analysis in Nude Mice
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Ten female BALB/C nude mice aged 6 weeks (Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were maintained in specific‐pathogen‐free conditions and used for xenograft studies following approval by the Animal Care and Use Committee of Henan Provincial People's Hospital, People's Hospital of Zhengzhou University, People's Hospital of Henan University. For xenograft formation, we gave each mouse a total volume of 200 μl PBS containing 5 × 106 lentivirus‐transduced ECA109 cells by subcutaneous injection into the left flanks. Each group included five mice. Xenograft tumor growth rates were monitored weekly by evaluating volume under the use of the 0.5 × (length × width2) formula. Four weeks later, we collected the xenograft tumors from the mice. Paraffin‐embedded xenograft tumors were processed by immunohistochemistry using antibodies against ki‐67 (ab15580, 1:100; Abcam), Bax (33–6400, 1:300; Invitrogen), Bcl‐2 (MA5‐11757, 1:50; Invitrogen), MMP9 (ab76003, 1:1000; Abcam), and MMP2 (ab86607, 1:100; Abcam), biotinylated anti‐mouse or anti‐rabbit IgG second antibody (ab64255 or ab64256, 1:100, Abcam) and 3,3‐diaminobenzidine tetrahydrochloride solution (MedChemExpress), as described elsewhere.37
6
Immunoblotting Antibody Validation
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The anti-p65 antibody (sc-372), the anti-PDGFR-β antibody (sc-432), the anti-β-actin antibody (sc-517582) and the anti-NG2 antibody (sc-166251) were obtained from Santa Cruz Inc. The antibodies anti-CD54 (ICAM-1) (555511), anti-CD62E (E-selectin) (551145), anti-CD106 (VCAM-1) (555647) and IgG1-κ Isotype Control (555749) were purchased from BD Biosciences (Heidelberg, Germany). Peroxidase-labeled anti-rabbit antibody (NIF 824) and peroxidase-labeled anti-mouse antibody (NIF 825) were obtained from GE Healthcare (Freiburg, Germany). The anti-HO-1 antibody (ADI-SPA-895) was from Enzo Life Sciences (Lörrach, Germany). The anti-α-tubulin antibody (ab56676), the anti-myeloperoxidase (MPO) antibody (ab9535), the anti-ICAM-1 antibody (ab124760), the anti-8-OHDG antibody (ab10802), the anti-eNOS antibody (ab5589), the anti-VCAM-1 antibody (ab134047), the anti-CD68 antibody (ab1252212) and the secondary biotinylated goat anti-rabbit antibody (ab64256) were purchased from Abcam.
7
Immunohistochemical Analysis of Ki-67 and SGTB
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8
Immunohistochemical Detection of RAGE and ACS
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After dewaxing and dehydration, tissue sections were blocked with 3.5% goat serum in PBS and stained with a rabbit polyclonal antibody against RAGE (Abcam, Cambridge, MA, catalog number ab3611) or ACS (Novus Biologicals, Littleton, CO, catalog number NBP1-78977) overnight at 4°C, followed by a 1-hour incubation with biotinylated goat anti-rabbit-IgG (Abcam, Cambridge, MA, catalog number ab64256) at room temperature. Staining was revealed by streptavidin peroxidase, developed with diaminobenzidine and counterstained with haematoxylin. Staining with PBS in place of primary antibody was used as negative controls. The slides were then examined under Nikon Eclipse Ti-E inverted microscope at 40x magnification.
9
Colorectal Cancer Tissue Analysis Protocol
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In this study, we obtained 50 pairs of colorectal primary tumors and adjacent noncancerous colorectal tissues (~3 cm away from tumors) from CRC patients undergoing colorectal resection in the First Affiliated Hospital of Wenzhou Medical University. None of the patients received any conventional treatment before surgery. The clinical features of these patients were shown in Table 1 . We confirmed tumor tissues by staining for cell proliferation using the cell-cycle marker Ki67 with anti-Ki67 antibody (ab15580, 1:100 dilution; Abcam, Cambridge, UK), biotinylated goat anti-rabbit IgG secondary antibody (ab64256, 1:300 dilution; Abcam), and a 3,3-diaminobenzidine (DAB) Kit (Vector Laboratories, Peterborough, UK), which was processed by immunohistochemistry using standard method [31 (link)]. Human tissue samples were used to evaluate the levels of circ_0089153, miR-198, and SENP1. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University and informed consent was provided by all subjects.
Correlation between circ_0089153 expression and the clinical features of CRC patients (n = 50)
| Clinical feature | Circ_0089153 expression (n) | ||
|---|---|---|---|
| High | Low | P value | |
| Age | 0.9453 | ||
| ≤50 years | 13 | 10 | |
| >50 years | 15 | 12 | |
| Gender | 0.5356 | ||
| Female | 14 | 15 | |
| Male | 12 | 9 | |
| TNM stage | <0.0001 | ||
| I+ II | 5 | 20 | |
| III | 22 | 3 | |
| Tumor size (cm) | 0.0003 | ||
| ≤3 | 7 | 15 | |
| >3 | 23 | 5 | |
10
Immunohistochemical Analysis of Tumor Markers
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The collected tumor tissue samples were fixed in 4% paraformaldehyde for 24 h at 25°C, embedded in paraffin and sliced into thin sections (3–5 µm). After dewaxing and rehydration, the sections were incubated with 3% hydrogen peroxide at 25°C for 30 min to block the endogenous peroxidase activity, then treated with 5% bovine serum albumin (Thermo Fisher Scientific, Inc.) at 37°C for 30 min to block non-specific binding. Subsequently, the sections were incubated with primary antibodies against Ki67 (cat. no. ab15580: 1:500), CD133 (cat. no. ab278053; 1:1,000), and cleaved caspase3 (cat. no. ab32042; 1:1,000; all Abcam) overnight at 4°C. The sections were washed with PBS 3 or 4 times, incubated with the biotinylated secondary antibody (Abcam, ab64256, dilution: 1:1,000) at 37°C for 1 h, then treated with streptavidin horseradish peroxidase at 37°C for 20-30 min. Images were captured under a light microscope (Axiovert 200; Zeiss AG). Positive stained cells were calculated by Image J (V1.8.0, National Institutes of Health) from the average of four random fields of view.
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