T cells and tumor tissues were lysed with Radioimmunoprecipitation (RIPA) Lysis and Extraction Buffer (Thermo Fisher Scientific) and quantified with a BCA Protein Assay Kit (Thermo Fisher Scientific). Protein lysates were separated on a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific). The PVDF membrane was blocked in AquaBlock Blocking Buffer (EastCoast Bio, ME, USA) for 2 h, followed by overnight incubation at 4 °C with the following primary antibodies: anti-CD133 (1:1000, Abcam), anti-GPC3 (1:400, Abcam), anti-β-actin (1:5000, Abcam) and anti-CD3ζ (1:5000, Abcam). Unbound antibodies were washed away with Tris–HCl buffer containing Tween 20, and the PVDF membrane was then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam) for 50 min at room temperature. Blots were detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) and visualized with a ChemiDoc™ Touch Imaging System (BIO-RAD, CA, USA).
Anti gpc3
Manufactured by Abcam
Sourced in United Kingdom
Anti-GPC3 is a laboratory reagent used for the detection and analysis of GPC3 (Glypican-3) in various biological samples. GPC3 is a cell surface proteoglycan that plays a role in cell growth and development. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and flow cytometry to identify and quantify GPC3 expression.
Automatically generated - may contain errors
Lab products found in correlation
Anti ck19, by Abcam (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Abcam (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Abcam (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti cd133, by Abcam (1 mentions)
Anti cd3ζ, by Abcam (1 mentions)
Biotinylated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Dab substrate, by Agilent Technologies (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Anti tubulin, by Abcam (1 mentions)
Anti cd34, by Abcam (1 mentions)
Anti α sma, by Abcam (1 mentions)
Anti hgf, by Abcam (1 mentions)
20 mm glass bottom cell culture dishes, by NEST Biotechnology (1 mentions)
Goat anti rabbit mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti afp, by Abcam (1 mentions)
5 protocols using anti gpc3
1
Quantitative Protein Analysis of T Cells and Tumor Tissues
2
Immunohistochemical Analysis of GPC3
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ACHN and 786-O cells were seeded on coverslips in 24-well plates. The cells were washed with PBS twice and fixed with 4% paraformaldehyde for 30 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 30 min in the dark and, after washing in PBS, the non-specific proteins were blocked in 1% bovine serum albumin (BSA) for 1 h. The cells were incubated at 4°C overnight with rabbit polyclonal anti-GPC3 (5 μg/mL) (ABCAM, Cambridge, UK) diluted in 1% BSA. After washing, cells were incubated with the biotinylated secondary antibody (1:200) (Santa Cruz Biotechnology, California, USA), diluted in 1% BSA for 45 min at 37°C and then exposed to an HRP-conjugated streptavidin complex (Santa Cruz Biotechnology, CA, USA). The reactions were visualized using DAB substrate (Dako, Cambridge, UK) and the slides were counterstained with hematoxylin. Densitometric analyses of GPC3 were performed with an Axioshop II Microscope (Zeiss, Germany) using the Software Axiovision (Zeiss). For the analyses, eleven different fields from the coverslips were used and 15 points were analyzed. The values were obtained on an arbitrary scale.
3
Immunofluorescence Staining of PDTOs
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The original tumor tissues were stored in 10% formalin, paraffin-embedded, dehydrated, and sliced for staining. After the PDTOs were dissociated from the hydrogel, they were either smeared on glass slides for H&E staining or transferred to 20-mm glass bottom cell culture dishes (NEST) for immunofluorescence staining. The samples were then fixed with 4% paraformaldehyde for 30 min, washed three times with PBS, added with 100 μL 0.5% Triton X-100, and incubated at room temperature for 30 min. Subsequently, the samples were washed again three times with PBS and blocked with 5% bovine serum albumin (BSA)/PBS solution at 37 °C for 30 min. Next, anti-AFP (1:100, Abcam), anti-CD34 (1:200, Abcam), anti-HGF (1:50, Abcam), anti-CK19 (1:200, Abcam), anti-tubulin (1:200, Abcam), anti-GPC-3 (1:500, Abcam), and anti-α-SMA (1:200, Abcam) antibodies diluted in 0.1% BSA/PBS were added, followed by incubation overnight at 4 °C. Next, the samples were incubated with goat anti-rabbit/mouse secondary antibody (1:200, Thermo Fisher) in 0.1% BSA/PBS and preserved at 37 °C for 2 h after washing three times with PBS. Finally, DAPI was applied and the samples were incubated for another 10 min. Immunofluorescence imaging was performed on a Zeiss LSM 710 confocal microscope.
4
Immunohistochemical and Immunofluorescence Analysis
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The specimens were fixed with 4% paraformaldehyde (PFA) and embedded in paraffin after tissue processing. After deparaffinization, rehydration, and antigen retrieval, 2-µm-thick sections were blocked with 10% normal donkey serum for 1 h at room temperature and stained with primary antibodies. The following primary antibodies were used in immunohistochemical staining: anti-SDC4 (1:500; Proteintech, Wuhan, China) and anti-GPC3 (1:500; Proteintech, Wuhan, China). Biotinylated anti-rabbit (Beyond, Shanghai, China) antibody was used as secondary antibodies according to the manufacturer's protocol. 3,3N-Diaminobenzidine Tertrahydrochloride (Beyond, Shanghai, China) was used as a chromogen according to the manufacturer's protocol. For double-immunofluorescence analysis, sections were pretreated in the same way as for IHC. The following primary antibodies were used: anti-SDC4 (1:50; mouse; Santa Cruz Biotechnology) and anti-KRT19 (1:500; rabbit; Abcam); anti-GPC3 (1:500; rabbit; Abcam) and anti-ACTA2 (1:500; mouse; Abcam). All slides were incubated with primary antibody overnight, which was diluted with 10% normal donkey serum after 1-h serum blocking. After DAPI (4’6-diamidino-2-phenylindole) staining, images were acquired using an Olympus BX51 microscope.
5
Comprehensive Liver Cancer Research Protocol
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DMEM, Opti-MEM, fetal bovine serum (FBS), and the penicillin–streptomycin solution were purchased from GIBCO/Thermo Fisher Scientific (Waltham, MA, USA). The anti-Pten, anti-p53, anti-GPC3, anti-CK19, and anti-Ki67 antibodies were from Abcam (Abcam, Cambridge, UK). T7 ligase, Q5 high-fidelity DNA polymerase, and T7E1 were purchased from New England Biolabs (Ipswich, MA, USA) and the Plasmid Extraction Kit and Genome DNA Isolation Kit were from Omega (Washington DC, USA). The PCR Purification Kit was from Axygen (Union City, CA, USA) and the SuperCoreTM Biopsy Needle MCXS1815LX was obtained from Argon Medical Inc. (Washington DC, USA). The percutaneous ethanol injection therapy (PEIT) needle was purchased from Hakko Co., Ltd (Nagano, Japan) and the Etamsylate Injection (2 mL: 0.5 g) solution was from Huazhong Pharmaceutical Co., Ltd (Wuhan, Hubei, China). The Shu Mianning II Injection solution was obtained from Nanjing Agricultural University (Nanjing, Jiangsu, China). Alpha Fetal Protein Assay Kit, Cancer Antigen 125 Assay Kit, and Cancer Antigen 19-9 Assay Kit were purchased from Roche Diagnostics GmbH (Mannheim, Germany).
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