Oropharynx squamous cell carcinoma tissue microarray (TMA) # 3 sections were provided by the Wisconsin Head and Neck Cancer SPORE. This TMA section contains 525 cores from 107 oropharynx squamous cell carcinoma, both HPV positive and HPV negative carcinoma; each sample is represented in triplicate 0.6 mm cores. The cancer cores include 171 primary, 207 lymph node metastatic, 6 distant metastatic, and 141 recurrent cancer cores. The tissue microarray (TMA) section was deparaffinized and blocked with 5% Goat serum. Antigens were retrieved in boiling 10 mM citrate buffer for 20 min. Tissues were then washed and stained overnight at 4 ˚C with anti-K17 (Abcam 109725), anti-CD8 (BioRad MCA351GT), anti-E-Cadherin (Abcam ab231303). Tissues were washed and stained with secondary antibodies conjugated with Alexa 488, Alexa 546 and Alexa 647. Tissues were washed and stained with Hoechst Dye before mounting in ProLong™ Diamond Antifade Mountant. The stained TMA was scanned by Vectra Automated Quantitative Pathology Imaging System at 20x objective. Scanned images were analyzed using inForm software (PerkinElmer). The software was trained using nine scanned images to distinguish tumor compartment (marked by positive E-Cadherin staining) and stromal compartment (marked by negative E-Cadherin staining). Each fluorescence channel was then analyzed within each compartment.
Ab231303
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab231303 is a primary antibody produced in rabbit. It is a general purpose antibody that can be used in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ab92547, by Abcam (19 mentions)
Ab76011, by Abcam (16 mentions)
Ripa buffer, by Beyotime (9 mentions)
Ripa lysis buffer, by Beyotime (8 mentions)
E cadherin, by Abcam (7 mentions)
Ab8245, by Abcam (7 mentions)
Ab181602, by Abcam (7 mentions)
Ab137321, by Abcam (7 mentions)
Ab15580, by Abcam (7 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Ab98952, by Abcam (6 mentions)
Ab9485, by Abcam (6 mentions)
Ab205719, by Abcam (6 mentions)
Ab205718, by Abcam (6 mentions)
Ab16667, by Abcam (6 mentions)
Ab76057, by Abcam (5 mentions)
Ab8226, by Abcam (5 mentions)
Bca protein assay kit, by Beyotime (5 mentions)
Ab27568, by Abcam (4 mentions)
Ab49254, by Abcam (4 mentions)
73 protocols using ab231303
1
Oropharynx Squamous Cell Carcinoma Tissue Microarray
2
Western Blot Analysis of Epithelial-Mesenchymal Transition
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Radioimmunoprecipitation assay (RIPA) was utilized to extract total cellular protein content, after which a BCA assay (Beyotime, Shanghai, China) was used for protein quantification. Samples were then electrophoretically separated and transferred onto PVDF membranes (Millipore, MA, USA). Following blocking for 2 h with 5% nonfat milk, blots were examined with proper primary and secondary antibodies. The bands of protein were then discovered via enhanced chemiluminescence (ECL) and analyzed with ImageJ (NIH, MD, USA). Antibodies used in this study were as follows: E-cadherin (abcam, ab231303, 1 : 1000), N-cadherin (abcam, ab98952, 1 : 1000), Vimentin (abcam, ab92547, 1 : 1000), Slug (abcam, ab27568, 1 : 1000), Snail (abcam, ab216347, 1 : 1000), HRP Goat Anti-Rabbit IgG (H+L) (ABclonal, AS014, 1 : 4000), HRP Goat Anti-Mouse IgG (H+L) (ABclonal, AS003, 1 : 5000).
3
Western Blot Analysis of Apoptosis and EMT Markers
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RIPA buffer was used to extract total protein. Proteins were quantified using a BCA protein determination Kit (KeyGEN Biotech, Nanjing, China). 30 μg protein was separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% non-fat milk for 1 h. The incubation of blots was conducted with antibodies at 4 °C overnight: Bax (ab32503, 1:1000, Abcam, Cambridge, MA, USA), Bcl-2 (ab117115, 1:1000, Abcam), ILF3 (ab225626, 1:1000, Abcam), AURKA (ab108353, 1:1000, Abcam), E2F1 (ab4070, 1:500, Abcam), E-cadherin (ab231303, 1:1000, Abcam), Vimentin (ab92547, 1:1000, Abcam), PI3K (#3811, 1:1000, CST, Danvers, MA, USA), phosphorylated PI3K (p-PI3K, #4228, 1:1,000, CST), AKT (#9272, 1:1,000, CST), phosphorylated AKT (p-AKT, #9271, 1:1000, CST) or GAPDH (ab8245, ab9485, 1:5000, Abcam) and then with HRP-conjugated second antibody (#7074, 1:1000, CST). Protein bands were detected with ECL Plus reagent (Pharmacia, Piscataway, USA), and visualized using a Gel Imaging System. Bands were then quantified using ImageJ software (National Institutes of Health). The expression of GAPDH was used for data normalization.
4
Immunofluorescence Analysis of Cell-Cell Adhesion
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Cells were cultured in 24-well plates on coverslips for 24 hours, then coverslips were collected, and cells were fixed with 4% formaldehyde and permeabilized with 0.5% Triton X-100 at room temperature. Followed, cells were incubated with polyclonal anti-E-cadherin (abcam, ab231303, 1 : 300) and anti-N-cadherin (abcam, ab98952, 1 : 300), and then incubation with Cy3 or FITC-labeled secondary antibody (ABclonal, Wuhan, China). Cells were then stained with DAPI (Beyotime, Shanghai, China) for 5 min. Images were captured under a fluorescent microscope.
5
Western Blot Analysis of CNN3 Regulation
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MG-63 and Saos-2 cells infected with LV-shCNN3 or LV-NC were seeded (4 × 105 cells/well) in 6-well plates. After being cultured for 48 h, cells were harvested, and the total protein content was isolated with RIPA buffer. After protein quantification, 30 μg of total protein was used for western blotting, which was conducted according to the methods described previously [33 (link)]. The primary antibodies used in this study were purchased from Abcam (Cambridge, MA, USA) and their details are as follows: anti-CNN3 (1:2000, ab151427), anti-MMP9 (1:1500, ab76003), anti-VEGF (1:5000, ab52917), anti-vimentin (1:1000, ab92547), anti-E-cadherin (1:1000, ab231303), p-p38 (1:1000, ab45381), p38 (1:1000, ab32142), ERK1/2 (1:1000, ab115799), p-ERK1/2 (1:800, ab214362), and anti-GAPDH (1:5000, ab181602).
6
Western Blot Analysis of EMT Markers
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Cells (HPDE6-C7, CaPAN-1, BxPC-3, PANC-1, and AsPC1) were lysed via RIPA buffer (Beyotime, Shanghai, China) and protein was collected. Concentrations of total protein were quantified through a BCA Protein Assay Kit (Sigma-Aldrich). Equal amounts of each sample were separated via 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transmembrane to polyvinylidene fluoride (PVDF) membranes (Merck, Darmstadt, Germany). Immediately, the membranes were incubated with primary antibodies overnight at 4°C, which were FBXL7 (ab59149, Abcam; 1.25 μg/ml), E-Cadherin (ab231303, Abcam; 1 μg/ml), N-cadherin (ab18203, Abcam; 1 μg/ml), Vimentin (ab137321, Abcam; 1:3,000), Snail 1 (ab53519, Abcam; 3 μg/ml), Snail 2 (ab82846, Abcam; 1:500), ZEB 1 (ab155249, Abcam; 1:3,000), Twist (ab49254, Abcam; 2.5 μg/ml), and GAPDH (ab181602, Abcam; 1:10,000). Goat anti-Human IgG/HRP (Solarbio, SE101) was employed as secondary antibody to incubate the membranes. Finally, Positive signals were detected with an ECL luminescence reagent (Sangon Biotech, Shanghai, China).
7
Immunohistochemical Analysis of Xenograft Specimens
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Paraffin specimens of harvested xenografts were cut into 4-μm-thick sections and mounted on silanized slides. The immunohistochemical staining was performed as published (13 (link)). The antibodies used were anti-Ki67 (Abcam, ab15580), anti-E-cadherin (Abcam, ab231303), anti-vimentin (Abcam, ab92547) and goat anti-rabbit secondary antibody (Abcam, ab205719). Finally, 3,3-diaminobenzidine was applied to visualize the results, and photographs were taken under a microscope (×100 and ×400).
8
Immunohistochemical Analysis of Tumor Biomarkers
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Tissues were embedded in paraffin, then sectioned into 4 μm thick slides, deparaffinized in xylene, rehydrated in a graded series of alcohol, and blocked with goat serum. After being stained with primary antibodies at 4°C overnight, sections were washed thrice then incubated with the secondary antibody for 20 min. After that, sections were reacted with 3,3′‐diaminobenzidine and counterstained with hematoxylin. Images were obtained by microscopy. Normal and tumor tissues were scored by evaluating their staining percentages. All the antibodies were listed as follows: anti‐Ki67 (ab15580, Abcam), anti‐E‐cadherin (ab231303, Abcam), anti‐SLUG (ab128485, Abcam), anti‐c‐MYC (ab32072, Abcam), anti‐BMI1 (ab126783, Abcam), and Goat anti‐Rabbit IgG H&L (ab205718, Abcam).
9
Protein Expression Analysis by Western Blot
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According to RIPA lysis buffer (Solarbio), total proteins were prepared. Next, 30‐μg protein samples were size‐fractionated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. After transferring onto PVDF membranes (2 h, 100 V), primary antibodies (4°C, overnight) were incubated. After incubation with secondary antibody, protein bands were observed according to the enhanced chemiluminescence solution (Vazyme, Nanjing, China). Antibodies (Abcam, Cambridge, UK) contains E‐cadherin (ab231303; 1:1500), N‐cadherin (ab98952; 1:1000), HOXB7 (ab152454; 1:1500), β‐actin (ab8227; 1:2000), and secondary antibodies (ab205718/ ab205719; 1:4000). Western blot assay was conducted at least three times.
10
Immunofluorescent Characterization of Cell Markers
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Cell fixation was performed using 4% paraformaldehyde in PBS at RT for 5 min. Except for the cells intended for surface antigen staining, the remaining cells were treated with PBS containing 0.1% Triton X-100 at room temperature for 5 min. To block nonspecific antibody binding sites, the cells were preincubated with PBS containing 5% goat serum at room temperature for 30 min. Primary antibodies (vimentin, Galectin-3, N-Cadherin, and E-Cadherin) were then applied to the cells and incubated at room temperature for 1 h. The primary antibody dilutions used were as follows: vimentin (1:1000, Abcam, (ab92547)), galectin-3 (1:1000, Abcam (ab227249)), N-cadherin (1:1000, Abcam, (ab18203)), and E-cadherin (1:1000, Abcam (ab231303)). After washing, the cells were incubated with goat anti-rabbit IgG H&L secondary antibody at room temperature for 1 h, followed by incubation with an anti-fluorescence attenuation mounting agent. No negative or positive controls were included in the study.
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