NEFM Antibody / Neuronal Cytoskeletal Marker Antibody (FY13056)

NEFM Antibody Mouse Brain IF. Immunofluorescent staining of FFPE mouse brain tissue using NEFM Antibody. Heat-induced epitope retrieval was performed in pH 8.0 EDTA buffer prior to incubation with the primary antibody at 5 ug/mL overnight at 4°C. A Cy3-conjugated goat anti-rabbit secondary antibody (red) was used for detection, and nuclei were counterstained with DAPI (blue). Bright red fluorescence highlights neuronal cell bodies and an extensive network of neuronal processes throughout the brain tissue, consistent with the expected distribution of neurofilament medium chain within the neuronal cytoskeleton. These results demonstrate that NEFM Antibody, a Neuronal Cytoskeletal Marker Antibody, specifically detects endogenous neurofilament medium chain in FFPE mouse brain tissue.

Western blot analysis of NF-M/NEFM using anti-NEFM antibody. Electrophoresis was performed on a 8% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human 293T whole cell lysates, Lane 2: human SH-SY5Y whole cell lysates, Lane 3: rat brain tissue lysates, Lane 4: mouse brain tissue lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA 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-NEFM antibody at 0.5 ug/ml overnight at 4oC, 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 was developed using an ECL Plus Western Blotting Substrate. A major band is detected at ~150 kDa, higher than the predicted molecular weight of 102 kDa. This migration pattern is well documented for NEFM and reflects its high degree of phosphorylation and extended coiled-coil structure, both of which cause slower electrophoretic mobility. The ~150 kDa band therefore represents the mature, post-translationally modified form of NEFM commonly observed in neuronal cells.

NEFM Antibody Mouse Brain IHC. Immunohistochemical staining of FFPE mouse brain tissue using NEFM Antibody. Heat-induced epitope retrieval was performed in pH 8.0 EDTA buffer prior to incubation with the primary antibody at 2 ug/mL overnight at 4°C. An HRP-conjugated goat anti-rabbit secondary antibody with DAB chromogen was used for visualization. Diffuse cytoplasmic staining is observed throughout neurons in the brain parenchyma, consistent with the expected distribution of neurofilament medium chain within the neuronal cytoskeleton. These results demonstrate that NEFM Antibody, a Neuronal Cytoskeletal Marker Antibody, specifically detects endogenous neurofilament medium chain in FFPE mouse brain tissue.

Immunohistochemical staining of NF-M/NEFM using anti-NEFM antibody. NF-M/NEFM was detected in a paraffin-embedded section of rat brain 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 ug/ml rabbit anti-NEFM antibody overnight at 4oC. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using an HRP secondary and DAB substrate.

NEFM Antibody Rat Brain IHC. Immunohistochemical staining of FFPE rat brain tissue using NEFM Antibody. Heat-induced epitope retrieval was performed in pH 8.0 EDTA buffer prior to incubation with the primary antibody at 2 ug/mL overnight at 4°C. An HRP-conjugated goat anti-rabbit secondary antibody with DAB chromogen was used for visualization. Widespread cytoplasmic staining is observed in neurons throughout the brain, with prominent labeling of neuronal cell bodies and processes, consistent with the expected distribution of neurofilament medium chain within the neuronal cytoskeleton. These results demonstrate that NEFM Antibody, a Neuronal Cytoskeletal Marker Antibody, specifically detects endogenous neurofilament medium chain in FFPE rat brain tissue.

NEFM Antibody Rat Cerebellum IHC. Immunohistochemical staining of FFPE rat cerebellum tissue using NEFM Antibody. Heat-induced epitope retrieval was performed in pH 8.0 EDTA buffer prior to incubation with the primary antibody at 2 ug/mL overnight at 4°C. An HRP-conjugated goat anti-rabbit secondary antibody with DAB chromogen was used for visualization. Prominent cytoplasmic staining is observed in Purkinje neurons and their processes at the interface between the molecular and granular layers, consistent with the expected distribution of neurofilament medium chain within the neuronal cytoskeleton. These results demonstrate that NEFM Antibody, a Neuronal Cytoskeletal Marker Antibody, specifically detects endogenous neurofilament medium chain in FFPE rat cerebellum tissue.

NEFM Antibody Mouse Cerebellum IHC. Immunohistochemical staining of FFPE mouse cerebellum tissue using NEFM Antibody. Heat-induced epitope retrieval was performed in pH 8.0 EDTA buffer prior to incubation with the primary antibody at 2 ug/mL overnight at 4°C. An HRP-conjugated goat anti-rabbit secondary antibody with DAB chromogen was used for visualization. Strong cytoplasmic staining is observed in cerebellar neurons, including prominent labeling of Purkinje cells and their processes, consistent with the expected distribution of neurofilament medium chain within the neuronal cytoskeleton. These results demonstrate that NEFM Antibody, a Neuronal Cytoskeletal Marker Antibody, specifically detects endogenous neurofilament medium chain in FFPE mouse cerebellum tissue.

NEFM Antibody SH-SY5Y Cell IP. Immunoprecipitation of NEFM from SH-SY5Y whole cell lysate using NEFM Antibody, followed by Western blot detection with the same antibody. Lane 1: Input SH-SY5Y whole cell lysate (30 ug). Lane 2: Rabbit IgG control immunoprecipitation. Lane 3: NEFM Antibody immunoprecipitation from 500 ug SH-SY5Y whole cell lysate using 2 ug of antibody. A specific band is detected at approximately 160 kDa, migrating above the predicted molecular weight of approximately 102 kDa due to the extensive phosphorylation characteristic of neurofilament medium chain. These results demonstrate that NEFM Antibody, a Neuronal Cytoskeletal Marker Antibody, specifically immunoprecipitates endogenous NEFM from human neuronal cells.

NEFM Antibody Human 293T Cell FACS. Flow cytometric analysis of fixed and permeabilized human 293T cells using NEFM Antibody. Cells were fixed with 4% paraformaldehyde, permeabilized, blocked with normal goat serum, and incubated with NEFM Antibody at 1 ug per 1 × 10^6 cells for 30 minutes. A DyLight 488-conjugated goat anti-rabbit secondary antibody was used for detection. Red: unstained cells. Green: rabbit IgG isotype control. Blue: NEFM Antibody. The clear rightward shift of the blue histogram relative to the isotype control demonstrates specific intracellular detection of endogenous neurofilament medium chain in human 293T cells. These results show that NEFM Antibody, a Neuronal Cytoskeletal Marker Antibody, is suitable for flow cytometric analysis of intracellular NEFM expression.

Immunohistochemical staining of NF-M/NEFM using anti-NEFM antibody. NF-M/NEFM was detected in a paraffin-embedded section of mouse brain 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 ug/ml rabbit anti-NEFM antibody overnight at 4oC. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using an HRP secondary and DAB substrate.

Flow cytometry analysis of fixed and permeabilized human 293T cells with NEFM antibody at 1ug/million cells (blocked with goat sera); Red=cells alone, Green=isotype control, Blue= NEFM antibody.