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

Activated JNK and p38

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® JNK pAb, Rabbit, (pTPpY ) (Cat.# V7931) and Anti-ACTIVE® p38,  Rabbit, (pTGpY ) (Cat.# V1211) were tested for labeling applications in tissue sections. Both pAbs have previously been demonstrated to function well in Western blots and cell culture staining. However, there is very little data on their utility in fixed tissue sections. In these experiments, the Abs demonstrated very specific labeling of various cellular subsets in rat brain hippocampal fixed/frozen sections. The protocol below worked in this model system but may need to be optimized by the end user.

Anti-ACTIVE® JNK and Anti-ACTIVE® p38 pAbs were tested on adult rat brain that was fixed, frozen and sectioned on a freezing/sliding microtome (30 microns). Anti-ACTIVE® p38 pAb (Figure 1, Panel A) appears to label cells lining the pyramidal cell layers (white arrows). Anti-ACTIVE® JNK pAb Figure 1, Panels B and C) appears to label cells resembling astrocytes. The labeling is specific to only a small portion of astrocytes and is often seen on cells closely associated with other cells or blood vessels. Note the "perinuclear" ring of label on the cells in Figure 1, Panels B and C (green arrow). Also note the label associated with processes coming from these cells (white arrows).

p38fig1A_small.jpg (11796 bytes)

p38fig1B_small.jpg (8385 bytes)

p38fig1C_small.jpg (7916 bytes)

Figure 1. Labeling of activated p38 and JNK in adult rat brain. Panel A: Cells surrounding pyramidal layer using Anti-ACTIVE p38 pAb (Cat.# V1211). Panels B and C: Astrocyte-like cells labeled using Anti-ACTIVE JNK pAb (Cat.# V7931).

This procedure was performed in 48 well tissue culture plates by moving the 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 sections three times, 10 minutes each, in PBS.
  4. Block 90 minutes in 5% donkey serum + 0.01% Triton® in PBS.
  5. Incubate overnight at 4°C in Anti-ACTIVE® JNK (Cat.# V7931) and Anti-ACTIVE® p38 (Cat.# V1211) pAbs diluted 1:500 in 1% donkey serum +  0.01% Triton® X-100 in PBS.
  6. Wash sections three times, 10 minutes each, in PBS.
  7. Incubate in donkey anti-rabbit Cy™3 ( Jackson ImmunoResearch ) diluted 1:1,000 in PBS.
  8. Wash sections 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
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