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The Art of IHC: Immunohistochemistry with Anti-ACTIVE® Antibodies
Activated MAPK
Anti-ACTIVE® MAPK (pTEpY) (Cat.# V8031) and Anti-ERK 1/2 (Cat.# V1141) pAbs were tested for labeling applications in
tissue sections. Both pAbs have previously been demonstrated to function well in Western
blots while Anti-ACTIVE® MAPK has applications in staining cultured cells
(ICC). In these experiments, the Abs demonstrated very specific labeling of various
cellular subsets in rat brain. Anti-ACTIVE® MAPK was also tested on mouse
brain.
This experiment demonstrated that these antibodies have applications in fixed tissue
immunohistochemistry. The 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.
Anti-ACTIVE® MAPK and Anti-ERK 1/2 pAb were tested on adult rat brain that
was fixed, frozen and sectioned on a freezing/sliding microtome (30 microns). Anti-ACTIVE®
MAPK appeared to label both neurons (Figure 1, Panel A, white arrows) and astrocytes
(Figure 1B, green arrows) in the hippocampal sections. Note the typical appearance of
astrocytes surrounding the blood vessel in Figure 1, Panel B. Compare the morphology of
these cells to the ones labeled with Anti-GFAP pAb (Cat.# G5601) an
astrocyte specific marker) in an adjacent section (Figure 1, Panel C, green arrows).
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Figure 1. Labeling of activated MAPK in
hippocampal sections from adult frozen rat brain. Panel A:
Anti-ACTIVE® MAPK pAb (Cat.# V8031) labeled
section. Labeling appears to be specific to astrocytes in this area of the hippocampus.
Note the typical astrocyte morphology of the Anti-ACTIVE® MAPK positive cells
(white arrows) surrounding the blood vessel (yellow arrows). Anti-ACTIVE® MAPK
also labeled neurons in the hippocampal molecular layer (data not shown). Panel B:
Astrocytic cells are also labeled with Anti-ERK 1/2 pAb (Cat.# V1141) in an
adjacent section. Anti-ERK 1/2 pAb labels both phosphorylated and nonphosphorylated forms
of MAPK. These astrocytes (white arrows) are also seen in close association with a blood
vessel (yellow arrows). Panel C: Anti-GFAP pAb (Cat.# G5601); glial
acidic protein, an astrocyte specific marker) labeling (white arrows) in an adjacent
section shows typical astrocyte morphology. Only a subset of astrocytes is labeled with
Anti-ACTIVE® MAPK and Anti-ERK 1/2 pAb as demonstrated by the intense
Anti-GFAP label seen throughout the section (data not shown). Panel D:
Low power image of CA3 region stained with Anti-ACTIVE® MAPK pAb. Note the
absence of neuronal staining. Panel E: CA3 region labeled with Anti-ERK
1/2 shows a subset of pyramidal cells (arrows) not labeled with Anti-ACTIVE®
MAPK. These neurons are expressing MAPK but there is no detectable phosphorylation at this
stage. Panel F: DAPI staining of Panel E image. |
Since Anti-ERK 1/2 pAb is not specific to phosphorylated MAPK, it should at least label
the same cell types as Anti-ACTIVE® MAPK. In fact, Anti-ERK 1/2 pAb did label
astrocytes as Anti-ACTIVE® MAPK did (Figure 2, Panel A, green arrow) but much
more label was seen in neuronal subsets (Figure 2, Panel B). Note the highly specific
nature of the label in Figure 2, Panel B (white arrows): only a subset of these
hippocampal neurons are labeled. This is demonstrated by the DAPI double label in Figure
2, Panel C which shows the section is full of cells not positive for ERK. This sort of
heavy neuronal label was never seen in the sections stained with Anti-ACTIVE®
MAPK.
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Figure 2. Labeling of all forms of MAPK (activated and
nonactivated) in adult rat brain. Panel A: Neurons labeled using Anti-ERK 1/2 pAb
(Cat.# V1141).
Panel B: Astrocytes labeled using Anti-ERK 1/2 pAb pAb. Panel C: DAPI
staining. |
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® X-100 in PBS.
Incubate overnight at 4°C in Anti-ACTIVE®
MAPK (Cat.# V8031)
and Anti-ERK 1/2 (Cat.#
V1141) pAbs diluted 1:500 in 1% donkey serum + 0.01% Triton® in PBS.
Wash three times, 10 minutes each, in PBS.
Incubate in donkey anti-rabbit Cy™3
(Jackson ImmunoResearch) diluted 1:1,000 in PBS.
Wash three times, 10 minutes each, in PBS.
Mount in Vectashield® + DAPI.
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