Anti-neurofilament

The reaction of anti-neurofilament antibodies with high molecular weight aggregates from rat neuronal cytoskeletal proteins provided direct evidence for neurofilament cross-linking after 2,5-hexanedione administration (Lapadula et al. 1986). Immunoblotting with antibodies specific for phosphorylated forms of cytoskeletal proteins has demonstrated a reduction of phosphorylation in neurofilament proteins and microtubule-associated-protein 2 (MAP-2) after 2,5-hexanedione treatment (Abou-Donia et al. 1988). 

Anti-neurofilament marker, e.g., the monoclonal antibody 2H3 and anti-SV2 (Developmental Studies Hybridoma Bank, University of Iowa, see http //dshb.biology.uiowa. 

Presynaptic antigens that work well include a cocktail of anti-neurofilament (for instance the monoclonal antibody 2H3) and anti-SV2 to fully label both the axon and the nerve terminal. 

Law AJ, Harrison PJ. 2003. The distribution and morphology of prefrontal cortex pyramidal neurons identified using anti-neurofilament antibodies SMI32, N200 and FNP7. Normative data and a comparison in subjects with schizophrenia, bipolar disorder or major depression. J Psychiatr Res 37(6) 487-499. 

Trojanowski JQ, Lee VMY. Anti-neurofilament monoclonal antibodies Reagents for the evaluation of human neoplasms. Acta Neuropathol. 1983 59 155-158. 

Notest aData for compounds 3 and 4 taken from ref. 263 data for compound 10 taken from ref. 266. bRats with lesioned sciatic nerves were treated with the indicated compounds, and sacrificed on the 18th day of the experiment. Axonal diameter and cross-sectional area were quantitated by anti-neurofilament staining, and myelin levels were quantitated by myelin basic protein-immunoreactive stain density. 

To investigate neuronal synapses in the white matter of mouse spinal cord, immuno-cytochemistry with two P antibodies was performed. The mouse anti-p38 antibody labels synaptic vesicle protein, with a secondary labeled with a 543 fluorophore were previously used in the lab. The rabbit anti-neurofilament antibody labels axons cut in cross-section within the white matter, with a secondary labeled with a 488 fluorophore. 

The double-label experiment was performed, but no label was seen for the neu-rofllament with the 543 fluorophore (Fig. 14.7a, b, c. Problem experiment). The expected labeling for p38 was seen in the red channel but no neurofilament labeling was seen in the green channel. No 1° antibody control for each antibody (results not shown) confirmed that there was no labeling with mouse anti-p38 alone but samples with rabbit anti-neurofilament did not have labeling. 

Mouse anti-p38 dilution was 1 1000 and rabbit anti-neurofilament was 1 2000. Rinses with PBS contained 5% normal rabbit serum, 10 mg/ml of BSA. 

Fig. 14.9 Case No. 5 initial results. In single-antibody experiments, mouse spinal cord was incubated individually with the different 1° antibodies. The spinal cord has a central core called the gray matter containing neuronal cell bodies and an outer rim called the white matter containing myelinated axons and no neuronal cell bodies, (a) Labeling of the white matter with mouse anti-neurofilament antibody showed cross-sections of labeled axons as small dots, (b) Mouse anti-neurofilament antibody-labeled gray matter contained randomly oriented axons, (c) Mouse anti-HuC-labeled neuronal cell bodies and no labeling was found because no neuronal cell bodies are found in the white matter, (d) In the gray matter, the mouse anti-HuC-labeled the numerous neuronal ceU bodies...

Rabbit anti-neurofilament 1 2000 and mouse anti-HuC 1 500 were used. 

HuC 1° antibody was not specific for antigen - The control results showed that there was no neurofilament labeling with anti-HuC incubations alone in the white matter (Fig. 14.8c, d), suggesting the HuC antibody was not binding to the axons. The control results also showed that with anti-neurofilament alone (Fig. 14.8e, f), the same labeling was seen with both P antibodies together (Fig. 14.10a, b). This observation eliminates the mouse anti-HuC 1° antibody as a cause of the problem. 

Fig. 14.11 Case No. 5 correct experiment. The spinal cord white matter has only neuronal axons and no neuronal cell bodies. The following micrographs are pairs from double-label experiments showing the white matter, (a) The cells labeled with rabbit anti-neurofilament and goat anti-rabbit 488 fluorophore showed axons, (b) The same filed as a showed no labeling for HuC as expected, (c) Incubation with no rabbit anti-neurofilament and with mouse anti-HuC followed by both 2° antibodies showed no label for neurofilament as expected, (d) Same field as (c) with no label in the HuC channel for axons, (e) Incubation with rabbit anti-neurofilament and without mouse anti-HuC and both 2° antibodies showed the labeling for axons, (f) Same field as (e) with no labeling in the HuC channel...

Ksiezak-Reding H, Dickson DW, Davies P et al. (1987) Recognition of tau epitopes by anti-neurofilament antibodies that bind to Alzheimer neurofibrillary tangles. Proc Natl Acad Sci USA 84, 3410-3414. 

Askanas V, Alvarez RB, MirabeUa M et al. (1996) Use of anti-neurofilament antibody to identify paired-helical filaments in inclusion-body myositis. Ann Neurol 39, 389-391. 

Fig. 36. Immunocytochemical staining patterns of two monoclonal anti-bodies directed against non-phosphorylated and phosphorylated neurofilaments were studied in the cerebellum of developing normal rats. A. Non-phosphorylated neurofilaments on postnatal day 11. B. Day 21. Basket cell axons form a characteristic brush-like plexus around the initial segment of the Purkinje cell axon. C. Phosphorylated neurofilaments on postnatal day 13, D. Postnatal day 21. Stained filaments are restricted to Purkinje cell and basket cell axons and are absent from the Purkinje cell cytoplasm. Calibration bars in A and C 30/tm, in B and D 10/tm. Marc et al. (1986).

Blocking agents were incorrect - The blocking serum was 5% rabbit serum and the neurofilament ° was made in rabbit. Thus, the T antibody goat anti-rabbit 488 bound the rabbit IgG in the buffer before the solution was incubated with the tissue section. The T" antibody goat anti-rabbit 488 bound to the normal... 

Fig. 14.8 Case No. 4 explanation. The problem was that the blocking solution contained normal rabbit serum that reacted with an antibody, (a) Both of the 1° antibodies mouse anti-p38 and rabbit anti-NF (neurofilament) bound to the expected antigens, (b) The incubation solution contains normal rabbit serum IgG as a blocking protein. In 2° antibody incubations the goat anti-mouse 488 fluorophore antibody bound to the mouse anti-p38 antibody. However, the goat anti-rabbit 543 fluorophore bound to the rabbit IgG in the blocking serum and not to the rabbit anti-NF, as expected, (c) Changing the serum in the incubation solution to normal goat serum allowed both 2° antibodies to find to the appropriate 1° antibodies...

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