Anti-Nefh Antibody Picoband®

Western blot analysis of Nefh using anti-Nefh antibody (A05307).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: rat brain tissue lysates,
Lane 2: mouse brain tissue lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-Nefh antigen affinity purified polyclonal antibody (Catalog # A05307) at 0.25 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for Nefh at approximately 117-220KD. The expected band size for Nefh is at 117KD.

IHC analysis of Nefh using anti-Nefh antibody (A05307).
Nefh was detected in paraffin-embedded section of rat brain tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-Nefh Antibody (A05307) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

IHC analysis of Nefh using anti-Nefh antibody (A05307).
Nefh was detected in a paraffin-embedded section of human cerebellum tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-Nefh Antibody (A05307) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Flow Cytometry analysis of Neuro-2a cells using anti-Nefh antibody (A05307).
Overlay histogram showing Neuro-2a cells stained with A05307 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-Nefh Antibody (A05307, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

IHC analysis of Nefh using anti-Nefh antibody (A05307).
Nefh was detected in paraffin-embedded section of mouse spinal cord tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-Nefh Antibody (A05307) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

Images of immunofluorescence staining in each group. (A) Pictures represent the expression of NF-200, GAP-43 and MAP-2, respectively. Rats in each group were sacrificed at Day 14 after the MCAO. Scale bar = 50 μm. (B) Immunofluorescence analysis of NF-200 ( n = 3), ∗ p < 0.05, as compared with the AAV-empty group, # p < 0.05, as compared with the AAV-NT-1 group. (C) Immunofluorescence analysis of GAP-43 ( n = 3), ∗ p < 0.01, as compared with the AAV-empty group, # p < 0.01, as compared with the AAV-NT-1 group. (D) Immunofluorescence analysis of MAP-2 ( n = 3), ∗ p < 0.05, as compared with the AAV-empty group.
Index in PubMed under a CC BY license. PMID: 29487502

Effects of Netrin-1 overexpression and/or JNK inhibition on the axonal regeneration after the MCAO. (A) The representative images of western-blot analysis for NF-200, GAP-43 and MAP-2. Rats in each group were sacrificed at Day 14 after the MCAO. (B) Western-blot analysis of NF-200 ( n = 3), ∗ p < 0.01, as compared with the AAV-empty group, # p < 0.05, as compared with the AAV-empty group. (C) Western-blot analysis of GAP-43 ( n = 3), ∗ p < 0.05, as compared with the AAV-empty group, # p < 0.01, as compared with the AAV-NT-1 group. (D) Western-blot analysis of MAP-2 ( n = 3), ∗ p < 0.05, as compared with the AAV-empty group.
Index in PubMed under a CC BY license. PMID: 29487502