Deparaffinization and protein extraction of FFPE tissue was carried out using Qproteome FFPE Tissue Kit (Qiagen, Hilden, Germany; #1042481) following manufacturer’s instructions. Extracted proteins were determined by Bradford assay (BIO-RAD, CA, USA; #5000205). Quantified proteins were mixed with 5 × SDS-PAGE loading buffer (Biosesang, Seongnam, Korea; S2002) in concentration of 2 μg/μL and boiled at 95 °C for 5 min. Equal quantities of protein were separated to SDS-PAGE gel and transferred to nitrocellulose membranes (BIO-RAD; #1704158). Membranes were blocked by incubation in 5% skim milk in Tris-buffered saline (TBS) with 0.1% Tween-20 and probed with antibody against ATX (1:1000; Abcam, Cambridge, UK; ab140915), LPA1(1:2000; Abcam; ab166903), and LPA2(1:1000; Abcam; ab38322) diluted in 1% BSA in TBS, 0.1% Tween 20, and 0.02% NaN3. The membranes were washed and then incubated with secondary antibodies (HRP conjugated anti-mouse IgG, or anti-rabbit IgG) (1:20,000; Santa Cruz, TX, USA) for 1 h at room temperature. The bands were visualized using WesternBright ECL (Advansata, CA, USA; K-12045-D50) after washing the membrane and exposed to X-ray film.
Ab166903
Manufactured by Abcam
Sourced in United Kingdom
Ab166903 is a laboratory reagent. It is a recombinant antibody that can be used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Ab38322, by Abcam (2 mentions)
Ab92552, by Abcam (2 mentions)
Ab140915, by Abcam (1 mentions)
Qproteome ffpe tissue kit, by Qiagen (1 mentions)
Sds page loading buffer, by Biosesang (1 mentions)
Hrp conjugated anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
1 oleoyl lysophosphatidic acid sodium salt, by Bio-Techne (1 mentions)
Pd98059, by Thermo Fisher Scientific (1 mentions)
Sc 51907, by Santa Cruz Biotechnology (1 mentions)
Transwell culture plate, by Corning (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8, by Beyotime (1 mentions)
Male balb c nude mice, by Charles River Laboratories (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab8224, by Abcam (1 mentions)
Ab40772, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Chemiluminescence hrp substrate, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
6 protocols using ab166903
1
Extraction and Immunoblotting of Proteins from FFPE Tissue
2
Lysophosphatidic Acid Signaling Mechanisms
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Recombinant human ATX (also known as Ectonucleotide Pyrophosphatase/Phosphodiesterase-2) was purchased from R&D Systems, Inc., Minneapolis, MN, USA. 1-Oleoyl lysophosphatidic acid sodium salt was from Tocris Bioscience (Bristol, UK). 17-β estradiol (estrogen) was from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). PD98059 [the phosphorylated extracellular signal-regulated kinase (p-ERK) inhibitor] was from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). ATX antibody for western blotting was from Abcam (Cambridge, UK; ab77104) and for immunohistochemistry from Santa Cruz Biotechnology (Texas, USA; sc-374222). Antibodies against LPA receptors (LPA1, LPA2, LPA3) were from Abcam (ab166903, ab38322 and ab219267, respectively). Total/phosphorylated extracellular signal-regulated kinases (t/p-ERK) were from Cell Signaling Technology, Inc. Danvers, MA, USA (4695s and 4370s). Antibodies against GAPDH were from Santa Cruz Biotechnology (sc-51907). Crystal violet staining solution was from Tiangen Biotech Co., Ltd. (Beijing, China). Cell Counting kit (CCK)-8 was from Beyotime Institute of Biotechnology, Haimen, China (C0037). Transwell culture plates were from Corning Incorporated (Corning, NY, USA). Lipofectamine 2000™ transfection reagent was from Invitrogen, Thermo Fisher Scientific, Inc.
3
Xenograft Assay for Evaluating circLPAR3 in SCC25 Cells
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Animal experiments were approved by the Animal Ethics Committee of Changsha Stomatological Hospital. 10 BALB/c male nude mice (4 weeks old, 15–20 g) (Vital River, Beijing, China) were reared at specific pathogen‐free condition. For the xenograft assay, SCC25 cells (1 × 107) with sh‐circLPAR3#2 or sh‐NC were subcutaneously injected into the back of each nude mouse (5 mice per group). Tumor volume was recorded once a week [(length × width2)/2]. Twenty‐eight days later, the mice were sacrificed and their xenograft tumors were stripped. The obtained xenograft tumors were fixed with 4% formaldehyde and then embedded in paraffin. Immunohistochemistry (IHC) analysis was carried out as previously described.21 Primary antibodies included LPCAT1 (ab166903, Abcam) and PCNA (ab92552, Abcam).
4
Immunohistochemical Evaluation of EDG2 Expression
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Immunohistochemistry (IHC) staining was carried out as described previously [19 (link)]. Briefly speaking, 4 μm-thick specimen slides for IHC staining were deparaffinized, and peroxidase was quenched with methanol and 3% H2O2 for 15 min. All sections were boiled with pressure in citrate buffer for 3 min and blocked overnight at 4°C. After incubated for 4 h with primary rabbit monoclonal anti-human EDG2 antibody (ab166903, Abcam, UK) at 1:100 dilution, the slides were washed with phosphate buffered saline (PBS), incubated with HRP-conjugated goat anti-rabbit antibody (ab6721; Abcam, UK) for 15 min at 1:100 dilution, and washed again with PBS. The staining of the slides was performed with the avidin-biotin-peroxidase complex. Finally, the sections were visualized with diaminobenzidine and counterstained with hematoxylin.
The scoring system for IHC staining was presented previously [16 (link)]. Staining intensity was divided into four grades: 0, none; 1, weak; 2, moderate; 3, strong. The percentage of specifically positive staining tumor cells was classified with the following grades: 0 (<5%), 1 (6%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (>75%). The final score was expressed by multiplying the staining intensity and the percentage of specifically positive staining tumor cells.
The scoring system for IHC staining was presented previously [16 (link)]. Staining intensity was divided into four grades: 0, none; 1, weak; 2, moderate; 3, strong. The percentage of specifically positive staining tumor cells was classified with the following grades: 0 (<5%), 1 (6%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (>75%). The final score was expressed by multiplying the staining intensity and the percentage of specifically positive staining tumor cells.
5
Protein Expression Analysis Protocol
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Total protein was extracted using RIPA lysis buffer with protease and phosphates inhibitor (SA). 20 μg protein extracts were loaded onto each well of 10% SDS‐PAGE. Separated proteins were then transferred onto a PVDF membrane (Millipore). Following blocking with 5% skim milk powder, the membrane was probed with primary antibodies against PCNA at 1:1000 dilution (ab92552, Abcam), E‐cadherin at 1:10,000 dilution (ab40772, Abcam), Vimentin at 1:1000 dilution (ab92547, Abcam), Bax at 1:1000 dilution (ab32503, Abcam), Bcl‐2 at 1:1000 dilution (ab32124, Abcam), LPCAT1 at 1:1000 dilution (ab166903, Abcam), and β‐actin at a concentration of 1 μg/ml (ab8224, Abcam). Membranes were then incubated with an appropriate secondary antibody (Abcam). Protein bands were viewed using an enhanced chemiluminescence detection system with a chemiluminescence HRP Substrate (Millipore).
6
Human Sperm Collection and Culture
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Biggers, Whitten, and Whittingham (BWW) medium for human sperm collection and culture was prepared according to the fifth edition of the WHO Laboratory manual for the examination and processing of human semen and our previously published report (41 (link)). All saline components used in BWW medium and LPA (L7260) were purchased from Sigma-Aldrich (MO, USA). Anti-phosphotyrosine monoclonal antibody 4G10, anti-α-tubulin monoclonal antibody, and goat anti-mouse IgG–H&L chain-specific peroxidase conjugate were purchased from Merck Millipore Corporation (Billerica, MA, USA). Anti-p-PKA substrate antibody was purchased from Cell Signaling Technology Corporation (Danvers, MA, USA). Nifedipine, Verapamil, mibefradil, NNC 55-0369, β-cyclodextrin, and bovine serum albumin (BSA) were obtained from Sigma-Aldrich. JC-1 was from Molecular Probe (Eugene, OR). Anti-LPA1 antibody (ab166903), anti-LPA2 antibody (ab135980), anti-LPA3 antibody (ab23692), anti-LPA4 antibody (ab183076) and anti-LPA5 antibody (ab77680) were purchased from Abcam (MA, USA). Anti-LPA6 antibody (orb30731) was purchased from Biorbyt (UK).
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