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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).
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.
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Introduction | Activated MAPK | Activated JNK and p38 | Activated CaM KII and Activated Akt | Activated Caspase-3 and PARP p85 fragment |
