The cells were fixed in 1% paraformaldehyde for 30 min. After washing 3 times with PBS, the cells were blocked and permeabilized in blocking solution (PBS containing 3% bovine albumin and 0.2% Triton X-100) for 30 min at room temperature. The cells were then incubated with primary antibodies in blocking solution at 4 °C overnight, washed 3 times, and incubated with the corresponding secondary antibodies for 1 h at room temperature. The cells were washed twice and stained with DAPI (Sigma) for 5 min and then analyzed using a Leica DMI6000B microscope (Leica Microsystems). Primary antibodies used in this study were as follows: FoxA2 (R&D, AF2400, 1:200), Sox17 (RD, AF1924, 1:200), goat anti-Pdx1 (R&D, AF2419, 1:200), guinea pig anti-insulin (Dako, A056401, 1:300), and rabbit anti-C-peptide (Abcam, ab14181, 1:300).
Ab14181
Manufactured by Abcam
Sourced in United Kingdom
Ab14181 is a protein that binds to a specific target. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Normal goat serum (ngs), by Merck Group (2 mentions)
Fitc conjugated anti mouse igg, by Merck Group (2 mentions)
Ab8304 50, by Abcam (2 mentions)
Ab8304 100, by Abcam (2 mentions)
Dmi6000b microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Af1924, by R&D Systems (1 mentions)
Af2419, by R&D Systems (1 mentions)
Af2400, by R&D Systems (1 mentions)
Genepix 4400a microarray scanner, by Molecular Devices (1 mentions)
Yoyo 1, by Thermo Fisher Scientific (1 mentions)
A0566, by Agilent Technologies (1 mentions)
N5413, by Merck Group (1 mentions)
4 protocols using ab14181
1
Immunofluorescence Staining of Endoderm Markers
2
Immunostaining of Insulin-Producing Cells
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For immunostaining,29 the undifferentiated and differentiated cells were fixed and permeabilized. Normal goat serum (NGS, Sigma, G9023) was used to block the sites of nonspecific binding of primary antibodies. The primary antibodies in the present study were mouse monoclonal proinsulin + insulin (Abcam, ab8304-50), rabbit polyclonal anti-C peptide (Abcam, ab14181), and mouse monoclonal insulin receptor beta (Abcam, ab8304-100). Cy5.29-conjugated anti-rabbit IgG (Abcam, ab6564) and FITC-conjugated anti-mouse IgG (Sigma, F9137) were applied as secondary antibodies. The nuclei were counterstained by DAPI.
3
Single-Cell Analysis of Islet Cell Types
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Human islets were dispersed into single cells using trypsin and filtration. Single islet cells were loaded onto scWest chips (Protein Simple, San Jose, CA, USA), then lysed, electrophoresed and UV-crosslinked according to the manufacturer’s protocol. Chips were probed with primary antibodies against somatostatin (1:30, A0566; Dako, Denmark), insulin B chain (1:10, M093-3; MBL, USA) and insulin C-peptide (1:15, ab14181; Abcam, UK). The appropriate Alexa-conjugated secondary antibodies were used. DNA was stained with Yoyo1 (Invitrogen, USA). Immunofluorescence was detected with the GenePix 4400A microarray scanner (Molecular Devices, USA) at 2.5 μm resolution.
4
Immunofluorescence Staining of Pancreatic Cells
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The primary antibodies used in this study were: anti-nestin (Sigma, N5413), mouse monoclonal proinsulin+insulin, (Abcam, ab8304-50), rabbit polyclonal anti-C peptide (Abcam, ab14181) and mouse monoclonal insulin receptor beta (Abcam, ab8304-100) antibody. Cy5.29-conjugated antirabbit IgG (Abcam, Cambridge, USA, ab6564) and FITC-conjugated anti-mouse IgG (Sigma, St.
Louis, MO, F9137) were used as secondary antibodies. For immunoflourescence, the cells were cultured in six-well plates, fixed and permeabilized. They were then treated by normal goat serum (NGS, Sigma, G9023) to prevent nonspecific binding. Then, the cells were incubated with the primary antibody, and relevant secondary antibody. All of the antibodies were used at 1:1000 dilutions. Glycerol (70%) was used to mount the cover slips. Primary antibody was removed as negative controls for immunostaining.
Louis, MO, F9137) were used as secondary antibodies. For immunoflourescence, the cells were cultured in six-well plates, fixed and permeabilized. They were then treated by normal goat serum (NGS, Sigma, G9023) to prevent nonspecific binding. Then, the cells were incubated with the primary antibody, and relevant secondary antibody. All of the antibodies were used at 1:1000 dilutions. Glycerol (70%) was used to mount the cover slips. Primary antibody was removed as negative controls for immunostaining.
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