Immunofluorescent analysis of the neurofilament light chain in paraffin-embedded rat brain tissue (right) compared to a negative control without MBS608850 (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 RT. Tissues were then probed with MBS608850 in 0.3% BSA at a dilution of 1:20 for 1 hour at 37°C. Tissues were then incubated with an IgG (H+L) goat antimMouse secondary antibody, DyLight 488 conjugate for 1 hour at 37°C (green). Nuclei ( blue) were stained with DAPI. Images were taken at 40X magnification.
Immunofluorescent analysis of the neurofilament light chain in paraffin-embedded mouse brain tissue (right) compared to a negative control without MBS608850 (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 RT Tissues were then probed with MBS608850 in 0.3% BSA at a dilution of 1:20 for 1 hour at 37°C. Tissues were then incubated with an IgG (H+L) goat antimMouse secondary antibod, DyLight 488 conjugate for 1 hour at 37°C (green). Nuclei ( blue) were stained with DAPI. Images were taken at 40X magnification.
Immunofluorescent analysis of the neurofilament light chain in paraffin-embedded human brain tissue (right) compared to a negative control without MBS608850 (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 RT Tissues were then probed withMBS608850 in 0.3% BSA at a dilution of 1:20 for 1 hour at 37°C. Tissues were then incubated with an IgG (H+L) goat antimMouse secondary antibod, DyLight 488 conjugate for 1 hour at 37°C (green). Nuclei ( blue) were stained with DAPI. Images were taken at 40X magnification.
Immunohistochemistry analysis of the neurofilament light chain showing staining in the filaments of paraffin-embedded rat brain tissue (right) compared to a negative control without MBS608850 (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 RT, washed with ddH2O and PBS, and then probed with MBS608850 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.
Western Blot analysis of neurofilament, light chain was performed by loading 25ug of human and mouse brain tissue lysate per well onto a polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked. NF-L was detected at ~70kD using MBS608850 at a dilution of 0.5ug/ml in blocking buffer overnight at 4C, followed by a HRP-labeled secondary antibody for 1 hour at RT and detection with a chemiluminescent substrate.
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