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.
Ab8304 100
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3 protocols using ab8304 100
1
Immunostaining of Insulin-Producing Cells
2
Proinsulin and Insulin Receptor Immunostaining
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The following primary antibodies were used in this study: mouse monoclonal proinsulin+insulin, (Abcam, ab8304-50) and mouse monoclonal insulin receptor beta (Abcam, ab8304-100). FITC-conjugated anti-mouse IgG (Sigma, St. Louis, MO, F9137) was used as a secondary antibody. Cells were cultured in 6-well plates on a cover slip, fixed in 4% paraformaldehyde, and permeabilized with 0.3% Triton X-100 in PBS for 30 min at 37°C. Following three washes with PBS, the cells were incubated with 10% normal goat serum (Sigma, G9023) in PBS for 30 min. Afterwards, the cells were incubated with the relevant primary antibody for 60 min, followed by incubation with the secondary antibody for 30 min. Cover slips were mounted with 70% glycerol in PBS. Several controls for immunostaining were used, and the primary antibody was omitted.
3
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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