Journal: Experimental gerontology

Article Title: Age-related impairment of autophagy in cervical motor neurons

doi: 10.1016/j.exger.2020.111193

Figure Lengend Snippet: Methods for immunohistochemical measurements of LC3B and p62 expression in motor neurons. Low magnification, stitched image (A) of the spinal cord in cross-section, used to identify the spinal segment by comparison to standard atlases. Multicolor-labeled fluorescence image shows LC3B and p62 in the ventral horn of the cervical spinal cord at C4 predominately in the gray matter regions. Insert represents area used to obtain high magnification image in (B) and (C). LC3B and p62 fluorescence (B) was measured in motor neurons identified by ChAT immunoreactivity (C) using manually generated regions of interest (ROIs) in the maximum projection images. See Methods for details. ChAT, Choline-Acetyl Transferase. Scale bar in 200 μm (A) and 100 μm (B and C).

Article Snippet: When multiple bands were evidenced in a blot (LC3B and ATG5 antibodies), the available blocking peptides for LC3B (NB100-2220PEP, 1:100; Novus) and ATG5 (NB110-53818PEP, 1:100; Novus) were used to detect the specific bands of interest.

Techniques: Immunohistochemical staining, Expressing, Comparison, Labeling, Fluorescence, Generated

Journal: Experimental gerontology

Article Title: Age-related impairment of autophagy in cervical motor neurons

doi: 10.1016/j.exger.2020.111193

Figure Lengend Snippet: Expression of the autophagosome marker LC3 in the cervical spinal cord and motor neurons of mice across age groups. Representative blot (A). Bar graphs show mean ± 95% CI of individual protein expression measurements of LC3-I (B), LC3-II (C) and the LC3-II/I ratio (D) per animal. There were no age-related differences in the protein expression levels of these markers or in the LC3-II/I ratio in cervical spinal cord of mice across age groups. Representative high magnification image of LC3 and ChAT immunofluorescence in motor neurons of mice at 6-, 18-, and 24-months of age (E). Bar graphs show mean ± 95% CI of LC3 expression in motor neurons, averaged per section and animal (for details, see methods section) in putative phrenic motor neurons (F) and non-phrenic motor neurons (G). LC3B expression in putative phrenic motor neurons at 24-months of age was ~25% higher than both the 6- and 18-month old groups. There were no age-related differences in LC3B expression at non-phrenic motor neurons. Scale bar: 100 μm; *, statistically significant difference between groups, post hoc Tukey-Kramer HSD, p < 0.05.

Article Snippet: When multiple bands were evidenced in a blot (LC3B and ATG5 antibodies), the available blocking peptides for LC3B (NB100-2220PEP, 1:100; Novus) and ATG5 (NB110-53818PEP, 1:100; Novus) were used to detect the specific bands of interest.

Techniques: Expressing, Marker, Immunofluorescence

Journal: Experimental gerontology

Article Title: Age-related impairment of autophagy in cervical motor neurons

doi: 10.1016/j.exger.2020.111193

Figure Lengend Snippet: Correlation between LC3B and p62 relative fluorescence intensity in individual motor neurons in the cervical spinal cord of aging male and female mice. Each point represents an individual motor neuron, symbols represent animals and colors represent age groups. Z-scores were calculated using the mean and SD for all animals. Shaded areas show bivariate normal density ellipses (90% of distribution) for each age group. Male mice at 18-and 24-months of age displayed a shift to the upper left corner (impaired autophagy) when compared to the 6-month old group in both putative phrenic (A) and non-phrenic (C) motor neurons. Female mice displayed no differences across age groups in either putative phrenic (B) or non-phrenic (D) motor neurons.

Article Snippet: When multiple bands were evidenced in a blot (LC3B and ATG5 antibodies), the available blocking peptides for LC3B (NB100-2220PEP, 1:100; Novus) and ATG5 (NB110-53818PEP, 1:100; Novus) were used to detect the specific bands of interest.

Techniques: Fluorescence

Journal: Experimental gerontology

Article Title: Age-related impairment of autophagy in cervical motor neurons

doi: 10.1016/j.exger.2020.111193

Figure Lengend Snippet: Relative expression of the autophagy markers LC3 and p62 in individual cervical motor neurons. Data are presented as mean ± 95% CI (arbitrary units) across age, sex and motor neuron pool, and are relative to the background-subtracted gray matter immunofluorescence intensity.

Article Snippet: When multiple bands were evidenced in a blot (LC3B and ATG5 antibodies), the available blocking peptides for LC3B (NB100-2220PEP, 1:100; Novus) and ATG5 (NB110-53818PEP, 1:100; Novus) were used to detect the specific bands of interest.

Techniques: Expressing, Immunofluorescence