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The Art of IHC: Immunohistochemistry with Anti-ACTIVE® Antibodies

Activated Caspase-3 and PARP p85 Fragment pAbs

By Randy Hoffman
Promega Corporation

This article contains five sections:
Introduction | Activated MAPK | Activated JNK and p38 |
Activated CaM KII and activated Akt | Activated Caspase-3 and PARP p85 Fragment

Anti-ACTIVE® Caspase-3 pAb (Cat.# G7481) and Anti-PARP p85 Fragment pAb (Cat.# G7341) were tested for labeling applications in paraffin-embedded mouse brain sections.

This experiment demonstrated that these antibodies have applications in fixed tissue immunohistochemistry. Anti-ACTIVE® Caspase-3 pAb specifically labeled cells that were also shown by DAPI staining to have the condensed nuclear morphology typical of apoptotic cells. Similar results were seen for cells stained with the Anti-PARP p85 Fragment pAb.

Figure 5 PARP and caspase-3 smcopy.jpg (8995 bytes)

Figure 1. Demonstration of Anti-ACTIVE® Caspase-3 and Anti-PARP p85 Fragment pAb-positive cells in post natal day 0 (P0) mouse brain paraffin embedded sections. Three Anti-ACTIVE® Caspase-3 pAb (Cat.# G7481)-positive cells (green and red arrows) and  corresponding DAPI-stained nuclei (Panel B). Note the correspondence of Anti-ACTIVE® Caspase-3 pAb label in Panel A with the typical apoptotic condensed nuclear morphology in Panel B. The Anti-PARP p85 Fragment pAb (Cat.# G7341) positive cell in (Panel C, white arrow) corresponds to an apoptotic DAPI stained nucleus in (Panel D, white arrow).

 

Method for Staining Paraffin-Embedded, Postnatal Day 0 Mouse Brain Sections.  (All steps are performed at room temperature.)

  1. Embed tissue in paraffin after fixation in 4% paraformaldehyde. Six micron sections are used for this protocol. Note: Best results will be obtained if the animal is perfused with fix and postfixed after dissection.

  2. Deparaffinize by washing tissue times for 5 minutes each in Hemo De (Fisher Scientific) or xylene. Rinse tissue sections for 2 minutes in 100% ethanol. Transfer sections to 95% ethanol for 2 minutes, then transfer them to 70% ethanol for 2 minutes. Finally, rinse tissue sections 2 times for 2 minutes each in PBS.

  3. Permeabilize for 10 minutes in PBS + 0.1% Triton® X-100.

  4. Wash sections 2 times for 5 minutes each in PBS.

  5. Block endogenous peroxide activity by incubating sections in 0.3% H2O2 in PBS for 30 minutes.

  6. Wash sections 2 times for 5 minutes each in PBS.

  7. Block for 45 minutes in PBS + 5% Donkey serum.

  8. Incubate in Anti-ACTIVE® Caspase-3 pAb diluted 1:125 or Anti-PARP pAb diluted 1:50 in PBS + 1.0% donkey serum for 60 minutes.

  9. Wash sections 3 times for 5 minutes each in PBS.

  10. Incubate in biotin conjugated donkey anti-rabbit pAb (Jackson ImmunoResearch) diluted 1:500 in PBS for 60 minutes.

  11. Wash sections 3 times for 5 minutes each in PBS.

  12. Incubate in RTU (Ready To Use) ABC reagent (Vector Laboratories) for 60 minutes.

  13. Wash sections 3 times for 5 minutes each in PBS.

  14. Develop with DAB substrate kit (Vector Laboratories) for 10 minutes.

  15. Wash 3 times for 5 minutes each in water.

  16. Mount in Vectashield® + DAPI (Vector Laboratories).

Introduction | Activated MAPK | Activated JNK and p38 |
Activated CaM KII and activated Akt | Activated Caspase-3 and PARP p85 Fragment
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