- 13-1300 - Invitrogen Antibodies NEFH antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of neurofilament heavy chain was performed by loading 25 µg of mouse brain tissue lysate per well onto a polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked. NF-H was detected at ~180-220 kD using a neurofilament heavy chain antibody (Product # 13-1300) at a dilution of 2 µg/mL in blocking buffer overnight at 4C, followed by a HRP-labeled secondary antibody for 1 hour at room temperature and detection with a chemiluminescent substrate.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of NEFH was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR819005_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of NEFH was performed by loading 20 µg of SH-SY5Y wild type (Lane 1), SH-SY5Y CAS9 (Lane 2), SH-SY5Y NEFH KO (Lane 3) whole cell extracts. The blot was probed with Anti-NEFH Monoclonal Antibody (RMdO-20)(Product # 13-1300) using 0.5 µg/mL dilution and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177). Loss of signal upon CRISPR mediated knockout (KO) confirms that the antibody is specific to NEFH. An uncharacterized band was observed at ~42 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-NEFH Monoclonal Antibody (RMdO-20) (Product # 13-1300) and a 150 kDa band corresponding to NEFH was observed across SH-SY5Y and IMR-32. Whole cell extracts (30 µg lysate) of SH-SY5Y (Lane 1), IMR-32 (Lane 2), LNCaP (Lane 3) and MCF7 (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein 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 µg/mL) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of the neurofilament heavy chain in paraffin-embedded mouse brain tissue (right) compared to a negative control without primary antibody (left). Tissue sections were deparaffinized with xylene, and rehydrated with ethanol. To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0) and microwaved for 8-15 min. Following antigen retrieval, tissues were washed with water and PBS, and then blocked in 0.3% BSA for 30 min at room temperature. Tissues were then probed with a neurofilament heavy chain monoclonal antibody (Product # 13-1300) in 0.3% BSA at a dilution of 1:20 for 1 hour at 37°C. Tissues were then incubated with a Goat anti-Mouse IgG (H+L) Secondary Antibody, DyLight 488 conjugate for 1 hour at 37°C (green). Nuclei (blue) were stained with DAPI. Images were taken at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of the neurofilament heavy chain showing staining in the filaments of paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0) and microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Neurofilament H chain monoclonal antibody (Product # 13-1300) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of the neurofilament heavy chain showing staining in the filaments of paraffin-embedded rat brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0) and microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Neurofilament H chain monoclonal antibody (Product # 13-1300) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of the neurofilament heavy chain in paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). Tissue sections were deparaffinized with xylene, and rehydrated with ethanol. To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0) and microwaved for 8-15 min. Following antigen retrieval, tissues were washed with water and PBS, and then blocked in 0.3% BSA for 30 min at room temperature. Tissues were then probed with a neurofilament heavy chain monoclonal antibody (Product # 13-1300) in 0.3% BSA at a dilution of 1:20 for 1 hour at 37°C. Tissues were then incubated with a Goat anti-Mouse IgG (H+L) Secondary Antibody, DyLight 488 conjugate for 1 hour at 37°C (green). Nuclei (blue) were stained with DAPI. Images were taken at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of the neurofilament heavy chain in paraffin-embedded rat brain tissue (right) compared to a negative control without primary antibody (left). Tissue sections were deparaffinized with xylene, and rehydrated with ethanol. To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0) and microwaved for 8-15 min. Following antigen retrieval, tissues were washed with water and PBS, and then blocked in 0.3% BSA for 30 min at room temperature. Tissues were then probed with a neurofilament heavy chain monoclonal antibody (Product # 13-1300) in 0.3% BSA at a dilution of 1:20 for 1 hour at 37°C. Tissues were then incubated with a Goat anti-Mouse IgG (H+L) Secondary Antibody, DyLight 488 conjugate for 1 hour at 37°C (green). Nuclei (blue) were stained with DAPI. Images were taken at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of the neurofilament heavy chain in paraffin-embedded mouse brain tissue (right) compared to a negative control without primary antibody (left). Tissue sections were deparaffinized with xylene, and rehydrated with ethanol. To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0) and microwaved for 8-15 min. Following antigen retrieval, tissues were washed with water and PBS, and then blocked in 0.3% BSA for 30 min at room temperature. Tissues were then probed with a neurofilament heavy chain monoclonal antibody (Product # 13-1300) in 0.3% BSA at a dilution of 1:20 for 1 hour at 37°C. Tissues were then incubated with a Goat anti-Mouse IgG (H+L) Secondary Antibody, DyLight 488 conjugate for 1 hour at 37°C (green). Nuclei (blue) were stained with DAPI. Images were taken at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of the neurofilament heavy chain showing staining in the filaments of paraffin-embedded mouse brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0) and microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Neurofilament H chain monoclonal antibody (Product # 13-1300) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of the neurofilament heavy chain in frozen sections of human cortex. Tissues were probed with a neurofilament heavy chain monoclonal antibody (Product # 13-1300, red) at a dilution of 1:20, then incubated with a Alexa Fluor 594 Goat anti-Mouse IgG (H+L) Secondary Antibody. Nuclei (blue) were stained with DAPI. Data courtesy of Min Sun Kim at Seoul National University, Korea.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Effects of PRP on morphological recovery of regenerated nerves in rabbits with sciatic nerve injury . (A) Images of myelinated nerve fibers using toluidine blue staining. The percentage of myelinated nerve fibers in the medium- and high-concentration PRP groups was higher than that in the model and low-concentration PRP groups. Black arrows indicate myelinated nerve fibers. Scale bars: 20 mum. (B) Immunofluorescent staining of the NEFH (NF; green, stained by fluorescein isothiocyanate, white arrows). The presence of neurofilaments in the medium- and high-concentration PRP groups was significantly higher than that in the model and low-concentration PRP groups. Scale bars: 20 mum. (C) Myelin sheaths of regenerated nerves observed by transmission electron microscopy. The white arrows indicate myelinated nerve fibers. Scale bars: 2 mum. (D) Lamellar structure of myelin sheaths. The thickness of the myelin sheath in the medium- and high-concentration PRP groups was greater than that in the model and low-concentration PRP groups. Scale bars: 100 nm. (E-G) Quantitative results of the percentage of myelinated nerve fibers (E), integral optical density of the neurofilament (F), and thickness of the myelin sheath (G). Data in E and F are presented as means +- SD ( n = 7) and were analyzed by Tukey's multiple comparison test. Data in G are presented as median and 95% confidence interval ( n = 7) and were analyzed by Tukey's multiple comparison test. ** P < 0.01, *** P < 0.001, **** P

13-1300

Antibody data