The preparation that this antibody was raised against is dominated by axonal forms of NF-H which are heavily phosphorylated on the multiple repeated NF-H KSP type sequences, and this antibody reacts very strongly with these phosphorylated repeats. Reactivity with non-phosphorylated KSP sequences is orders of magnitude weaker.
Immunohistological analysis of a mouse hippocampus section stained with Anti-NF-H Antibody (1:2,000 | red) and Anti-MBP Antibody (A85322 | 1:5,000 | green). The blue is DAPI staining of nuclear DNA. Following transcardial perfusion with 4% paraformaldehyde, brain was post fixed for 24 hours, cut to 45µM, and free-floating sections were stained with the above antibodies. The Anti-NF-H Antibody labels a network of axons of different neurons, while the Anti-MBP Antibody stains myelin sheath around these axons.
Mixed neuron/glia cultures stained with Anti-NF-H Antibody (green) and Anti-NF-L Antibody (A85286 | red). Axons contain phosphorylated NF-H and NF-L so appear yellow, while dendrites and perikarya only contain NF-L and so appear red. DNA is shown in blue.
Figure 3: Western Blot - Anti-NF-H Antibody (A85336)
Western blot analysis of different tissue lysates using Anti-NF-H Antibody (1:10,000 | green): [1] protein standard (red), [2] rat brain, [3] rat spinal cord [4] mouse brain, and [5] mouse spinal cord lysate. Strong band at about 220kDa corresponds to the phosphorylated axonal form of the NF-H subunit. Smaller proteolytic fragments of NF-H are also detected with Anti-NF-H Antibody.
Figure 4: Western Blot - Anti-NF-H Antibody (A85336)
Western blot analysis of Anti-NF-H Antibody. Blot of 20 µg rat brain lysate was probed with Anti-NF-H Antibody (1:25,000). A prominent band at 200 kDa corresponds to phosphorylated form of NF-H.
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