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The Art of IHC: Immunohistochemistry with Anti-ACTIVE® Antibodies
Activated CaM KII and Activated Akt
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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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. |
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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.
- Remove brain and place in 4% paraformaldehyde overnight at
4°C.
- Freeze tissue and cut 30 micron sections on a
freezing/sliding microtome.
- Wash 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® 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.
- Wash three times, 10 minutes each, in PBS.
- Incubate in Jackson ImmunoResearch’s donkey
anti-rabbit Cy™3 diluted 1:1000 in PBS.
- Wash three times, 10 minutes each, in PBS.
- Mount in Vectashield® + DAPI.
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