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REACTIVITY SENSITIVITY MW (kDa)
H M R Endogenous 4
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Immunohistochemical analysis of paraffin-embedded human pancreas, showing staining of β-cells, using C-Peptide Antibody.

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Immunohistochemical analysis of paraffin-embedded mouse pancreas, showing staining of β-cells, using C-Peptide Antibody.

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Immunohistochemical analysis of paraffin-embedded rat pancreas showing staining of β-cells using C-Peptide Antibody.

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Immunohistochemical analysis of frozen mouse pancreas using C-Peptide Antibody.

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Confocal immunofluorescent analysis of mouse pancreas using C-Peptide Antibody (green). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

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Confocal immunofluorescent analysis of β-TC-6 cells (left) and HeLa cells (right) using C-Peptide Antibody (green). Actin filaments have been labeled with DY-555 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Immunohistochemistry (Paraffin)

A. Solutions and Reagents

  1. Xylene
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%)
  3. Deionized water (dH2O)
  4. Hematoxylin (optional)
  5. Wash Buffer:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
  6. Antigen Unmasking Citrate: 10 mM Sodium Citrate Buffer: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O7•2H2O) to 1 L dH2O. Adjust pH to 6.0.
  7. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
  8. Blocking Solution: TBST/5% normal goat serum (#5425): to 5 ml 1X TBST add 250 µl normal goat serum.
  9. Detection System: VECTASTAIN® Elite ABC, including biotinylated secondary antibody (Vector Laboratories).
  10. Substrate: Vector® NovaRED™ (Vector Laboratories).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

  1. Deparaffinize/hydrate sections:
    1. Incubate sections in three washes of xylene for 5 minutes each.
    2. Incubate sections in two washes of 100% ethanol for 10 minutes each.
    3. Incubate sections in two washes of 95% ethanol for 10 minutes each.
  2. Wash sections twice in dH2O for 5 minutes each.

C. Antigen Unmasking

For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0; maintain at a sub-boiling temperature for 10 min. Cool slides on bench top for 30 min.

D. Staining

  1. Wash sections in dH2O three times for 5 minutes each.
  2. Incubate sections in 3% hydrogen peroxide for 10 minutes.
  3. Wash sections in dH2O twice for 5 minutes each.
  4. Wash sections in wash buffer for 5 minutes.
  5. Block each section with 100-400 µl blocking solution for 1 hour at room temperature.
  6. Remove blocking solution and add 100-400 µl primary antibody diluted in blocking solution to each section. Incubate overnight at 4°C.
  7. Prepare ABC solution per manufacturer's recommendations.
  8. Remove primary antibody and wash section three times with wash buffer for 5 minutes each.
  9. Add 100-400 µl biotinylated secondary antibody, diluted in TBST per manufacturer’s recommendation, to each section. Incubate 30 minutes at room temperature.
  10. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
  11. Cover sections with 100-400 µl pre-mixed ABC solution as needed and incubate in a humidified chamber for 30 min at room temperature.
  12. Wash section three times with wash buffer for 5 min each.
  13. Prepare Vector® NovaRED™ per manufacturer's recommendations.
  14. Apply 100-400 µl substrate to each section and monitor closely. 5-15 minutes generally provides an acceptable staining intensity.
  15. If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
  16. Wash sections in dH2O two times for 5 minutes each.
  17. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 seconds each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
    3. Repeat in xylene, incubating sections two times for 10 seconds each.
  18. Mount coverslips.

posted June 2005

revised February 2008

protocol id: 310

Immunohistochemistry (Frozen)

A. Solutions and Reagents

  1. Xylene
  2. Ethanol (anhydrous denatured, histological grade 100% and 95%)
  3. Hematoxylin (optional)
  4. 20X Phosphate Buffered Saline (PBS) (9808):
    1. To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  5. Fixative: 3% formaldehyde.
    1. To prepare 3% formaldehyde add 18.75 ml 16% formaldehyde to 81.25 ml 1X PBS, mix.
  6. Wash Buffer: 1X Tris Buffered Saline (TBS).

    To prepare 1L 1X TBS add 100 ml 10X Tris Buffered Saline (#12498) to 900 ml dH2O, mix.

  7. Methanol/Peroxidase: To prepare, add 10 ml 30% H202 to 90 ml methanol. Store at -20°C.
  8. Blocking Solution: 1X TBS/0.3% Triton-X 100/5% normal goat serum (#5425). To prepare: add 500 µl goat serum and 30 µl Triton-X 100 to 9.5 ml 1X TBS.
  9. Detection System: VECTASTAIN Elite ABC, including biotinylated secondary antibody (Vector Laboratories).
  10. Substrate: Vector® NovaRED™ (Vector Laboratories).

B. Sectioning

  1. For tissue stored at -80°C: remove from freezer and equilibrate at -20°C for approximately 15 minutes before attempting to section. This may prevent cracking of the block when sectioning.
  2. Section tissue at a range of 6-8 µm and place on positively charged slides.
  3. Allow sections to air dry on bench for a few minutes before fixing (this helps sections adhere to slides).

C. Fixation

  1. Fix sections for 15 min at room temperature in 3% formaldehyde, followed by 5 min in methanol at -20°C (do not rinse in between). Proceed with staining immediately (Section D).

D. Staining

  1. Wash sections in wash buffer twice for 5 min.
  2. Incubate for 10 minutes at room temperature in 3% H202 diluted in methanol.
  3. Wash sections in wash buffer twice for 5 min.
  4. Block each section with blocking solution for one hour at room temperature.
  5. Remove blocking solution and add 100-400 µl primary antibody diluted in blocking solution to each section.
  6. Prepare ABC solution per manufacturer's recommendations.
  7. Add 100-400 µl biotinylated secondary antibody, diluted in TBST per manufacturer's recommendation, to each section. Incubate 30 min at room temperature.
  8. Remove secondary antibody solution and wash sections three times with wash buffer for 5 min each.
  9. Cover sections with 100-400 µl pre-mixed ABC solution as needed and incubate in a humidified chamber for 30 min at room temperature.
  10. Wash section three times with wash buffer for 5 min each.
  11. Prepare Vector® NovaRED™ per manufacturer's recommendations.
  12. Apply 100-400 µl substrate to each section and monitor closely. 5-15 min generally provides an acceptable staining intensity.
  13. If desired, counterstain sections in hematoxylin per manufacturer's instructions.
  14. Wash sections in dH2O two times for 5 min each.
  15. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 seconds each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
    3. Repeat in xylene, incubating sections two times for 10 seconds each.
  16. Mount coverslips.

posted January 2006

revised April 2006

protocol id: 331

Page

Immunofluorescence (Frozen)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer: (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425) to 9 ml 1X PBS) and mix well. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
  5. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

    NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.

  6. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Frozen/Cryostat Sections (IF-F)

  1. For fixed frozen tissue proceed with Immunostaining (Section C).
  2. For fresh, unfixed frozen tissue, please fix immediately, as follows:
    1. Cover sections with 4% formaldehyde dilute in 1X PBS.

      NOTE: Formaldehyde is toxic, use only in fume hood.

    2. Allow sections to fix for 15 minutes at room temperature.
    3. Aspirate liquid, rinse three times in 1X PBS for 5 minutes each.
    4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
  7. Rinse three times in 1X PBS for 5 min each.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised November 2013

protocol id: 151

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.
  5. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

  6. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.

    NOTE: Formaldehyde is toxic, use only in a fume hood.

  2. Allow cells to fix for 15 min at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
  7. Rinse three times in 1X PBS for 5 min each.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised November 2013

protocol id: 24

Product Usage Information

Application Dilutions
Immunohistochemistry 1:100
Immunofluorescence 1:400

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

C-peptide Antibody detects endogenous levels of total C-peptide protein.


Species Reactivity: Human, Mouse, Rat

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the sequence of human C-peptide. Antibodies are purified by protein A and peptide affinity chromatography.

Glucose homeostasis is regulated by hormones. Elevation of blood glucose levels during feeding stimulates insulin release from pancreatic β-cells through a glucose sensing pathway (1). Proinsulin, the insulin precursor molecule, is processed prior to its secretion. Insulin is composed of A-peptide and B-peptide which are joined by a disulfide bond. The center one-third of the molecule is cleaved and released as C-peptide, which has a longer half-life than insulin (2).


1.  Straub, S.G. and Sharp, G.W. (2002) Diabetes Metab. Res. Rev. 18, 451-463.

2.  Polonsky, K.S. and Rubenstein, A.H. (1984) Diabetes 33, 486-494.


Entrez-Gene Id 3630
Swiss-Prot Acc. P01308


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
DRAQ5® is a registered trademark of Biostatus Limited.