Immunofluorescent analysis of NEFL in mouse cerebellum  section. The mouse brain section was obtained following transcardial perfusion of the mouse with 4% paraformaldehyde, brain was post fixed for 24 hours, and cut to 45µM. Free-floating sections were stained using an NEFL polyclonal antibody (Product # PA1-10014) at a dilution of 1:5,000 as seen in red, and costained using a MBP polyclonal antibody (Product # PA1-10008) at a dilution of 1:5,000 as seen in green. The NEFL antibody labels dendrites and axons of neuronal cells, and the MBP antibody stains the network of myelin sheathes around axons.

Western blot was performed using Anti-NEFL Polyclonal Antibody (Product # PA1-10014) and a 68 kDa band corresponding to NEFL was observed in PC-12 differentiated to neurons, Mouse Cerebellum, Mouse Brain and Rat Brain but not in PC-12, Mouse Heart and Mouse Lung along with few uncharacterized bands (*). Membrane enriched extracts (30 µg lysate) of PC-12 (Lane 1), PC-12 differentiated to neurons (Lane 2), Mouse Cerebellum (Lane 3), Mouse Brain (Lane 4), Rat Brain (Lane 5), Mouse Heart (Lane 6) and Mouse Lung (Lane 7) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:15,000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). * - There were few uncharacterized bands observed in PC-12 differentiated to neurons, Mouse Cerebellum and Mouse Brain.