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The Art of IHC: Immunohistochemistry with Anti-ACTIVE® Antibodies
Activated JNK and p38
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).
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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.
- Remove brain and place in 4% paraformaldehyde overnight at
4°C.
- Freeze tissue and cut 30 micron sections on a
freezing/sliding microtome.
- Wash sections three times, 10 minutes each, in PBS.
- Block 90 minutes in 5% donkey serum + 0.01% Triton®
in PBS.
- 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.
- Wash sections three times, 10 minutes each, in PBS.
- Incubate in donkey anti-rabbit Cy™3 ( Jackson
ImmunoResearch ) diluted 1:1,000 in PBS.
- Wash sections three times, 10 minutes each, in PBS.
- Mount in Vectashield® + DAPI.
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