In Rat, Neurofascin has three observed isoforms , isoform 1 (138 kDa) which is known as Nfasc186, isoform 2 (133 kDa) and isoform 3 (132 kDa) which are both known as Nfasc155. These proteins are highly glycosylated and their observed molecular weight in Western blot is 186 kDa and 155 kDa respectively.
Mouse Neurofascin has a predicted molecular weight of 138 kDa (Swiss-Prot data) however the observed bands at 186 kDa and 155 kDa are thought to correspond to the glycosylated isoforms 1, 2 and 3 as observed in Rat (Sherman DL et al, 2005,PubMed:16337912)
ICC/IF image of ab31457 stained human SHSY5Y cells. The cells were 4% PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab31475, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Neurofascin antibody (ab31457)This image is courtesy of an abreview submitted by Carl Hobbs, King's College London, United Kingdom
Ab31457 used on Rat cerebellum, Purkinje cells are variously positive: there is a clear punctate positivity of secondary dendritic processes, a strong membraneous positivity of the somata and also a single strongly positive fibre that seemingly enters the Granule Cell Layer indicated by red arrowheads axons of P.cells.
The Glomeruli of the GCL are moderately positive.
There is also a linear pattern of positivity within the GCL that may well be fibres.
Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-Neurofascin antibody (ab31457)This image is courtesy of Sophie Pezet, CNRS, Paris, France
Immunohistochemistical detection of Neurofascin using antibody ab31457 on PFA perfusion-fixed rat brain sections. Primary antibody ab31457 was used at 1/1000 incubated for 18 hours @ 20°C in PBS + 0.3 % Triton X100. Secondary antibody: goat anti-rabbit Alexa Fluor 488 conjugated (1/1000). Immunostaining obtained in the rat hippocampus using this antibody diluted 1:1000. Sections come from a perfused-fixed animal. The immunostaining was performed using the `free floating` technique, using direct fluorescence. Some cytoplasmic staining was observed in granular neurons and neuronal processes (arrows on the figure on the right). The lefthand figure shows the hippocampal granular cell staining (objective X20), the righthand figure shows this at higher magnification. This staining is consistent with published observation of cytoplasmic localisation of Neurofascin in the cytoplasm of hippocampal cells and their axon initial segment (Burkarth
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Suzuki S et al. Spatio-temporal and dynamic regulation of neurofascin alternative splicing in mouse cerebellar neurons. Sci Rep7:11405 (2017).
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