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Immunofluorescence (Paraffin)

Confocal immunofluorescent analsyis of paraffin-embedded Rh30 xenograft, control (left) or rapamycin-treated (right), using PathScan® Apoptosis and Proliferation Multiplex IF Kit. Red = α-tubulin, green = phospho-Histone H3 (Ser10), and blue = cleaved PARP (Asp214).

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Immunofluorescence (Paraffin)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade, 100% and 95%.
  3. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  4. 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.
  5. 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
  6. 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.
  7. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Paraffin Sections (IF-P)

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


  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.
  4. Rinse sections twice in dH2O for 5 minutes each.

Antigen Unmasking:

NOTE: Consult product datasheet for specific recommendation for the unmasking solution.

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

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. Rinse three times in 1X PBS for 5 minutes each.
  2. Block specimen in Blocking Buffer for 60 minutes.
  3. While blocking, prepare primary cocktail by diluting as indicated on datasheet in Antibody Dilution Buffer.
  4. Aspirate blocking solution, apply diluted primary cocktail.
  5. Incubate overnight at 4°C.
  6. Rinse three times in 1X PBS for 5 minutes each.
  7. Prepare detection cocktail by diluting as indicated on datasheet in Antibody Dilution Buffer.
  8. Incubate 1–2 hours at room temperature in the dark.
  9. Rinse three times in 1X PBS for 5 minutes each.
  10. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
  11. For best results examine specimens immediately using appropriate excitation wavelengths. For long-term storage, store slides at 4°C protected from light.

posted July 2010

revised August 2011

Protocol Id: 445

Kit Includes

Products Included No. Volume Applicaton Dilution Reactivity Homology†
Primary Cocktail 7847 100 µl Immunofluorescence (Immunocytochemistry),
Immunofluorescence (Paraffin)‡
1:100 Human Monkey
Detection Cocktail 7843 100 µl Immunofluorescence (Immunocytochemistry),
Immunofluorescence (Paraffin)‡
1:100 N/A N/A
†Species predicted to react based on 100% sequence homology.
‡Immunofluorescence (Paraffin) protocol recommended unmasking buffer: Citrate
Kit Analytes Detection Dye Ex(max) (nm) Em(max) (nm)
Phospho-Histone H3 (Ser10) Alexa Fluor® 488 495 519
Cleaved-PARP (Asp214) Alexa Fluor® 647 650 665
α-Tubulin Alexa Fluor® 555 555 565

Product Description

The PathScan® Apoptosis and Proliferation Multiplex IF kit offers a novel method to simultaneously monitor mitotic index and programmed cell death using manual immunofluorescence microscopy, or automated imaging and laser scanning high content platforms. This kit contains a cocktail of three high quality primary antibodies targeted against α-tubulin, phospho-histone H3 (Ser10), and cleaved PARP (Asp214), as well as a detection cocktail utilizing the Alexa Fluor® series of fluorescent dyes. Antibody and dye pairings have been pre-optimized, and each kit contains enough reagents for 100 assays (based on a working volume of 100 μL/test).

Specificity / Sensitivity

α-Tubulin antibody detects endogenous levels of total α-tubulin protein. Phospho-Histone H3 (Ser10) antibody detects endogenous levels of histone H3 only when phosphorylated at Ser10. This antibody does not cross-react with other phosphorylated or acetylated histones. Cleaved-PARP (Asp214) detects endogenous levels of the large fragment (89 kDa) of human PARP1 protein produced by caspase cleavage. This antibody does not recognize full length PARP1 or other PARP isoforms.

Species Reactivity: Human

Source / Purification

Monoclonal antibodies were produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser10 of human histone H3, a synthetic peptide corresponding to residues surrounding Asp213 of human PARP, or with full-length chicken α-tubulin purified from brain extracts.

Apoptosis is a regulated physiological process leading to cell death. Initiator caspases cleave and activate downstream effector caspases that in turn cleave cytoskeletal and nuclear proteins like PARP, α-fodrin, DFF, and lamin A, which induce apoptosis (1). Cleavage of the nuclear poly (ADP-ribose) polymerase PARP occurs between Asp214 and Gly215, which separates the PARP amino-terminal DNA binding domain (24 kDa) from the carboxy-terminal catalytic domain (89 kDa) (2,3). PARP helps to maintain cell viability by playing key roles in many cellular processes, including DNA replication, repair, and recombination. Cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis (4). Cell proliferation can be measured by studying the phosphorylation state of histone H3 and microtubule assembly during mitosis. Histone H3 is one of four distinct core histone proteins found in the nucleosome. Phosphorylation of histone H3 at Ser10, Ser28, and Thr11 is tightly correlated with chromosome condensation during both mitosis and meiosis (5-7). Heterodimers composed of α-tubulin and β-tubulin form globular tubulin subunits common to all eukaryotic cells. Tubulin polymers known as microtubules are fundamental cytosolic fibers important in mediating cellular movement, including meiotic/mitotic chromosome alignment, cytoplasmic membrane vesicle transport, and nerve-cell axon migration (8).

1.  Nicholson, D.W. (1999) Cell Death Differ 6, 1028-42.

2.  Lazebnik, Y. A. et al. (1994) Nature 371, 346-347.

3.  Westermann, S. and Weber, K. (2003) Nat Rev Mol Cell Biol 4, 938-47.

4.  Nicholson, D.W. et al. (1995) Nature 376, 37-43.

5.  Oliver, F.J. et al. (1998) J. Biol. Chem. 273, 33533-33539.

6.  Hendzel, M.J. et al. (1997) Chromosoma 106, 348-60.

7.  Goto, H. et al. (1999) J Biol Chem 274, 25543-9.

8.  Preuss, U. et al. (2003) Nucleic Acids Res 31, 878-85.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.

PathScan® Apoptosis and Proliferation Multiplex IF Kit