For ICC, cells were seeded on a glass coverslip and cultured with control medium or CM from MIApaca‐2, BxPC‐3, SW1990 or PANC‐1 for 24 h. Cells were washed and fixed. Triton‐X‐100 was used to permeabilize the cells. After treating with 3% hydrogen peroxide, cells were incubated with antibodies against α‐SMA (ab32575, 1:100) and PDGFR‐α (ab203491, 1:100) from Abcam overnight, and horseradish peroxidase‐linked secondary antibodies for 1 h. Diaminobenzidine (DAB) was used for detection. For IF, cells were treated as in ICC and incubated with antibodies against α‐SMA (ab32575, 1:100), E‐cadherin (ab219332, 1:100), fibroblast activation protein (FAP, ab53066, 1:100) and epithelial cell adhesion molecule (EpCAM) (ab218448, 1:100) from Abcam overnight. The cells were then incubated with the secondary antibodies, Alexa Fluor 488 (A32731, Invitrogen) and Alexa Fluor 594 (A48271, Invitrogen). Subsequently, the coverslips were fixed and imaged.
Ab219332
Manufactured by Abcam
Sourced in United Kingdom
Ab219332 is a lab equipment product. It is a recombinant protein that can be used as a research tool. No further details are available.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Ab18197, by Abcam (2 mentions)
Sc 2005, by Santa Cruz Biotechnology (2 mentions)
Bca kit, by Beyotime (2 mentions)
Ab137321, by Abcam (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Ab203491, by Abcam (1 mentions)
Ab53066, by Abcam (1 mentions)
Ab32575, by Abcam (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay (ripa), by Thermo Fisher Scientific (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab214424, by Abcam (1 mentions)
Ab53519, by Abcam (1 mentions)
Ab27568, by Abcam (1 mentions)
Ab76011, by Abcam (1 mentions)
Ab51032, by Abcam (1 mentions)
Ab8277, by Abcam (1 mentions)
5 protocols using ab219332
1
Immunocytochemistry and Immunofluorescence Assay for Pancreatic Cancer Cell Lines
2
Western Blot Analysis of EMT and Angiogenesis Markers
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Hep3b cells were washed in PBS and lysed using the protein extraction reagent RIPA (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration of proteins was measured by BCA kit (cat. no. ab102536; Abcam). Equivalent amounts of proteins (30 µg) from each sample were electrophoresed on SDS-polyacrylamide gel (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane, blocked in 4% skim milk for 2 h at room temperature and incubated with the following specific primary antibodies: E-cadherin antibody (ab219332 1:1,000; Abcam), α-catenin antibody (ab51032 1:2,000; Abcam), N-cadherin (ab76011, 1:5000 dilution, Abcam), vimentin (ab92547 1:1,000; Abcam), p-PI3K (ab182651 1:1,000; Abcam), PI3K (ab227204 1:1,000; Abcam), p-AKT (ab38449, 1:500 dilution, Abcam), AKT (ab18785 1:1,000; Abcam), VEGF (ab214424 1:1,000; Abcam), VEGFR2 (ab221679 1:1,000; Abcam), Snail (ab53519 1:1,000; Abcam), Slug (ab27568 1:1,000; Abcam) and MPP9 (ab38898 1:1,000; Abcam) overnight at 4°C. β-actin (ab8277 1:1,000; Abcam) was used as internal reference. Then, the membranes were incubated in HRP-linked goat anti-rabbit IgG secondary antibody (ab97051; 1:10,000; Abcam) for 2 h at room temperature. Immunoreactivity was visualized by a colorimetric reaction using an ECL substrate buffer (EMD Millipore) and membranes were scanned with Gel Doz EZ imager (Bio-Rad Laboratories, Inc.).
3
Western Blot Analysis of Cell Line Proteins
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Total protein sample was extracted from cell lines with RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Proteins of equal amounts (30 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies against PCNA (1:1000, ab18197, Abcam), CDK4 (1:1000, ab226474, Abcam), E-cadherin (1:1000, ab219332, Abcam), N-cadherin (1:1000, ab76059, Abcam), and GAPDH (1:5,000; ab8245; Abcam) overnight at 4 °C. After an incubation with horseradish-peroxidase-conjugated secondary antibody (1:5000, SC-2005, Santa Cruz, Inc.) for 2 h at room temperature, the protein bands were visualized with the enhanced chemiluminescence (ECL) Plus kit (Beyotime Institute of Biotechnology).
4
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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The protein samples from cells or renal tissues were extracted using RIPA lysis buffer (Beyotime, Shanghai, China), and the concentration of protein was determined using a BCA kit (Beyotime). The protein samples, equivalent in amount, were separated using SDS-PAGE and subsequently transferred onto PVDF membranes (Millipore, Billerica, USA). The PVDF membranes were blocked with 5% fat-free milk for 2 h at room temperature. Subsequently, they were incubated overnight at 4°C with specific primary antibodies against α-SMA (1:1000, 19245, Cell Signaling Technology), E-cadherin (1:1000, ab219332, Abcam), vimentin (1:1000, ab137321, Abcam), Lin28A (1:1000, ab175352, Abcam), and GAPDH (1:10000, ab181602, Abcam). Following this, the membranes were washed and incubated with a horseradish peroxidase-conjugated secondary antibody (1:2000, sc-2005, Santa Cruz Biotechnology, Inc.) for 2 h at 20°C-25°C. The antibodies mentioned earlier were obtained from Abcam (Cambridge, UK). Immunoreactive bands were visualized using an enhanced chemiluminescence reagent ECL detection kit (Thermo Fisher Scientific, Inc.) and quantified using ImageJ software (National Institutes of Health, Bethesda, MD).
5
Western Blot Analysis of Protein Biomarkers
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Extraction of total protein sample was performed with radio-immuno-precipitation assay (Beyotime, Nanjing, Jiangsu, China). After protein quantification by a BCA kit (Beyotime Biotechnology), equal amount of protein (30 µg) was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred into polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Afterwards, the membranes were blocked with 5% skimmed milk in tris buffered saline containing 0.1% tween-20 (TBST) for 2 hours at room temperature, and incubated with specific primary antibodies against RHBDD1 (1 : 1000, HPA013972, Sigma), PCNA (1 : 500, ab18197, Abcam, Cambridge, United Kingdom), E-cadherin (1 : 1000, ab219332, Abcam), N-cadherin (1 : 1000, ab18203, Abcam), vimentin (1 : 1000, ab137321, Abcam), and GAPDH (1 : 5000, 10494-1-AP, Proteintech) at 4°C overnight, followed by incubated with horseradish peroxidase-conjugated secondary antibodies (#7074; 1 : 5000, Cell Signaling Technology) for 2 hours at room temperature. The immuno-reactive proteins were detected through an enhanced chemiluminescent detection system (Thermo Fisher Scientific).
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