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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Mouse IgG2a
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Confocal immunofluorescent analysis of Hep G2 (left) or HeLa (right) cells using AFP (3H8) Mouse mAb (Alexa Fluor® 555 Conjugate) (red). Actin filaments were labeled with Alexa Fluor® 488 phalloidin (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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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 goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. 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. 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. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  7. 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: 182

Product Usage Information

Application Dilutions
Immunofluorescence 1:50

Storage: Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Specificity / Sensitivity

AFP (3H8) Mouse mAb (Alexa Fluor® 555 Conjugate) recognizes endogenous levels of total AFP protein.


Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a recombinant fragment of human AFP.

Product Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 555 fluorescent dye and tested in-house for direct immunofluorescent analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated AFP (3H8) Mouse mAb #3903.


Alpha-fetoprotein (AFP) is a 65 kDa glycoprotein found in the mammalian fetal liver, yolk sac, and GI tract. While AFP expression in adult cells is low, it is aberrantly expressed in adult liver cancer cells (1,2). The tumor suppressor gene p53 and β-catenin are both involved in the regulation of AFP expression. In normal adult cells, p53 binds to the repressor region of the AFP gene, thereby blocking transcription. Mutations in both p53 and β-catenin are associated with aberrant expression of AFP. Research studies have shown that elevated serum AFP levels are predictive of hepatocellular carcinoma (3).


1.  Warnes, T.W. and Smith, A. (1987) Baillieres Clin Gastroenterol 1, 63-89.

2.  Peng, S.Y. et al. (2004) Int J Cancer 112, 44-50.

3.  Meier, V. et al. (2006) Comp Hepatol 5, 2.


Entrez-Gene Id 174
Swiss-Prot Acc. P02771


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.
The Alexa Fluor® dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.