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

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