For fluorescent microscopy, the cultured cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilised with 0.1% Triton X-100 for 10 min. After blocking for 30 min with 5% bovine serum albumin, the washed cells were incubated for 1 h at room temperature or overnight at 4°C with a primary antibody against the POU domain, class 5, transcription factor 1 (OCT4) (1:200, ab19857, Abcam), Transcription factor SOX-2 (SOX2) (1:200, ab92494, Abcam), Homeobox protein NANOG (NANOG) (1:100, ab109250, Abcam), Tra-1-60 (1:150, MAB4360, Merck), Tubulin β-3 (TUBB3) (1:500, ab18207, Abcam), smooth muscle alpha actin (SMA) (1:400, A2547, Merck) or α-fetoprotein (AFP) (1:200, ab3980, Abcam). Subsequently, the cells were incubated with fluorescence-labelled secondary Alexa Fluor 594 or 488 (1:1000, A11001, A11008 and A11012, Invitrogen) at room temperature for 1 h, protected from light. The cells were further incubated with 1 µM DAPI for nuclear staining. Between incubations, samples were washed with PBS containing 0.1% Triton X-100. Alkaline Phosphatase Live Stain 500X from Thermo Fisher Scientific was used for determining the enzyme activity following the manufacturer's instructions. Pictures were acquired using a Floyd Cell imaging station (Life Technologies).
Ab3980
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab3980 is a primary antibody product from Abcam. It is a polyclonal antibody raised against a specific antigen. The core function of this product is to bind and detect the target antigen in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
A11012, by Thermo Fisher Scientific (1 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
Ab19857, by Abcam (1 mentions)
A2547, by Merck Group (1 mentions)
Mab4360, by Merck Group (1 mentions)
Ab92494, by Abcam (1 mentions)
Ab18207, by Abcam (1 mentions)
Ab109250, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence reagent kit, by Merck Group (1 mentions)
Ab134045, by Abcam (1 mentions)
Ab125011, by Abcam (1 mentions)
Anti gapdh, by Beyotime (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Ab181602, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Lsm 700, by Zeiss (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
6 protocols using ab3980
1
Immunofluorescence Analysis of Stem Cell Markers
2
Exosomal Protein Profiling by Western Blot
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Protein samples extracted from cells using the Radio Immunoprecipitation Assay (RIPA Beyotime, Nantong, China) buffer were electrophoresed on 10% SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis), and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking in 5% skim milk for 2 h, membranes were incubated with anti-GAPDH (Beyotime, Nantong, China), anti-TSG101 (ab125011, Abcam), anti-CD63 (ab134045, Abcam) and anti-AFP (ab3980, Abcam) at 4oC overnight. 24 h later, membranes were incubated with secondary anti-body (Beyotime, Nantong, China) for 1 h. Band visualization was conducted using the enhanced chemiluminescence reagent kit (Millipore, Billerica, MA, USA).
3
Quantification of Serum AFP Levels
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Serum AFP levels were measured using ELISA (no. ab3980; Abcam, Cambridge, UK), and dilutions of excessively high AFP concentration, setup, adjustments and quality controls were performed according to the manufacturer’s instructions. The assay employed anti-human AFP antibody coated onto a 96-well plate. Standard or serum samples were pipetted into the wells and AFP present in samples was bound to the wells by the immobilized antibody. The wells were washed and biotinylated anti-human AFP antibody was added. Following washing of unbound biotinylated antibody, horseradish peroxidase-conjugated streptavidin was pipetted into the wells. The wells were washed again, TMB substrate solution was added, and color developed in proportion to the amount of bound AFP. Absorbance value was measured at 450 nm. AFP concentration in serum was achieved according to standard curves.
4
Western Blot Analysis of Protein Expression
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Western blot analysis was used to detect protein expression. Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate, pH 7.5) containing 1 mmol/L phenylmethyl sulfonyl fluoride for 30 minutes on ice. The samples were centrifuged, and the protein concentrations for each sample were determined by using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). The protein samples (20 µg) were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and then electrotransferred to a nitrocellulose membrane. After blocking with 5% nonfat dry milk in Tween-Tris buffered saline solution (0.1% Tween-20 in 100 mmol/L Tris–HCl [pH 7.5], 0.9% NaCl), the membranes were incubated with mouse monoclonal antibody against human AFP (ab3980, Abcam, USA) at a dilution of 1:1000 at 4 °C overnight. After washing, the blots were incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit IgG antibodies at a dilution of 1:10,000 for 1 hour. The blot was washed and developed by using chemiluminescence (Santa Cruz Biotechnology, Santa Cruz, USA). For the control, we detected glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with primary rabbit monoclonal antibodies against GAPDH (ab181602, Abcam, USA).
5
Immunostaining of Primary Liver Cells
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To validate the primary cells, cells were fixed with 4% paraformaldehyde (Biosesang, Korea) for 10 min at room temperature, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in Dulbecco’s phosphate-buffered saline (DPBS; Welgene) for 30 min at room temperature, and then washed three times with DPBS. The following primary antibodies were used: goat monoclonal anti-human serum albumin (A80-229A, 1:250, Bethyl laboratories, TX, USA), mouse monoclonal anti-human Hep-Par 1(OCH1E5, 1:250, Cell Marque, Darmstadt, Germany), and mouse monoclonal anti-alpha fetoprotein (ab3980, 1:250, abcam, Waltham, MA, USA). Samples were incubated with the primary antibodies for 16 h at 4 °C and then washed for 10 min three times with DPBS. The secondary antibodies used for staining were donkey anti-mouse IgG conjugated with Alexa® Fluor 488 (A-21202, Invitrogen, Eugene, OR, USA) and donkey anti-goat IgG conjugated with Alexa® Fluor 647 (A32849, Invitrogen). Samples were then incubated with secondary antibodies for 1 h at room temperature in the dark and washed for 10 min five times with DPBS. For nuclei staining, cells were incubated with DAPI (D9542-10MG, Sigma-Aldrich, St.Louis, MO, USA) for 10 min at room temperature in the dark and washed with PBS twice quickly. All fluorescence images were obtained using the LSM 700 (Zeiss, Oberkochen, Germany).
6
Immunofluorescence Analysis of Stem Cell Markers
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The cells were fixed in 4% paraformaldehyde solution (PanEco, Moscow, Russia) for 10 min at room temperature; washed with PBS; pre-incubated in 0.25% Triton X-100 and 1% BSA in PBS for 30 min; and incubated with primary antibodies to βIII tubulin (ab7751, Abcam, Cambridge, UK), S100b (ab52642, Abcam, Cambridge, UK), NANOG, SSEA4, OCT4, TRA-1-81, SOX2 (StemLight Pluripotency Antibody Kit, Cell Signaling Technology, Beverly, MA, USA), pancytokeratin (ab7753, Abcam, Cambridge, UK), desmin (ab32362, Abcam, Cambridge, UK), α-fetoprotein (ab3980, Abcam, Cambridge, UK), Nestin (ab105389, Abcam, Cambridge, UK) or PAX6 (ab5790, Abcam, Cambridge, UK) at +4 °C overnight. Secondary antibodies (Alexa Fluor 555 or Alexa Fluor 488 conjugated, Invitrogen, Carlsbad, CA, USA) were applied for 60 min in the dark. The nuclei were counterstained with DAPI (Sigma-Aldrich, St. Louis, MO, USA) solution (1 µg/mL in PBS). The signals were observed, and images were recorded with an Axio Observer.D1 inverted fluorescence microscope equipped with AxioCam HRc camera (Carl Zeiss, Oberkochen, Germany).
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