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

Activated CaM KII and Activated Akt

By Randy Hoffman
Promega Corporation

The 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® CaM KII pAb, Rabbit, pT286 (Cat.# V1111) and Anti-pS473 Akt (Cat.# G7441) pAbs were tested for labeling applications in tissue sections.

This experiment demonstrated that these antibodies have applications in fixed tissue immunohistochemistry. Both antibodies gave distinct labeling patterns on various cellular subsets. The protocols below worked in this model system and may need to be optimized by the end user. However, the methods presented below should be a good starting point for tissues processed in this fashion.

Anti-ACTIVE® CaM KII and Anti-pS473 Akt pAbs were tested on adult rat brain that was fixed, frozen and sectioned on a freezing/sliding microtome (30 microns). Anti-ACTIVE® CaM KII appeared to label strictly neurons (Figure 1, Panels A and B). Differences could be seen in neuronal labeling patterns depending on the hippocampal location, demonstrating the antibody's ability to label specific subsets of cells. Figure 1, Panels A and B demonstrate the heavy staining of neuronal cell bodies (white arrows) and streaming dendrites (green arrowheads).

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CaM_KIIfig1B_small.jpg (16209 bytes)

Figure 1. Labeling of activated CaM KII in adult rat brain. Panel A: Neurons labeled using Anti-ACTIVE® CaM KII pAb (Cat.# V1111). Panel B: Alternate plane section of labeling as in Panel A.

 

Figure 4 Akt smcopy.jpg (3445 bytes)

Figure 2. Frozen hippocampal sections from adult rat brain stained with Anti-S473 Akt pAb (Cat.# G7441) Panel A: Low power image from the CA4 region showing staining specific for dentric termini (arrows). Panel B: Higher power image showing dentate granule cell staining in both the cell body (white arrows) and the streaming dendrites (yellow arrows).

This procedure was performed in 48 well tissue culture plates by moving the tissue section from well to well.

Preparing Adult Rat Brain for IHC.

  1. Remove brain and place in 4% paraformaldehyde overnight at 4°C.
  2. Freeze tissue and cut 30 micron sections on a freezing/sliding microtome.
  3. Wash three times, 10 minutes each, in PBS.
  4. Block 90 minutes in 5% donkey serum +  0.01% Triton ® X-100 in PBS.
  5. Incubate overnight at 4°C in Anti-ACTIVE® CAM KII pAb (Cat.# V1111) diluted 1:500 and Anti-pS473 Akt (Cat.# G7441) diluted 1:50 in 1% donkey serum + 0.01% Triton® X-100 in PBS.
  6. Wash three times, 10 minutes each, in PBS.
  7. Incubate in Jackson ImmunoResearch’s donkey anti-rabbit Cy™3 diluted 1:1000 in PBS.
  8. Wash three times, 10 minutes each, in PBS.
  9. Mount in Vectashield® + DAPI.

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