Anti-Neurofascin antibody

• IHC-FoFr

Collaborator

Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-Neurofascin antibody (AB31457)

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

This image is courtesy of Sophie Pezet, CNRS, Paris, France

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Neurofascin antibody (AB31457)

ab31457 staining Neurofascin in Rat Primary Neurons DIV14 cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab31457 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• WB

Project

Western blot - Anti-Neurofascin antibody (AB31457)

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)

All lanes:

Western blot - Anti-Neurofascin antibody (ab31457) at 1 µg/mL

All lanes:

Brain (Mouse) Whole Cell Lysate at 10 µg

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 150 kDa

Observed band size: 120 kDa,155 kDa,186 kDa,55 kDa

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