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. 2016 Jun 8;75(7):673–688. doi: 10.1093/jnen/nlw040

FIGURE 6.

FIGURE 6.

Autophagosome analysis in capillary endothelium of the ventral horn cervical spinal cord in rats 7 and 30 days after tMCAO. (A) Immunofluorescence staining of spinal cord in control rats for Beclin-1 (green, arrowheads) (a, b) and LC3B (red, arrowheads) (a′, b′) showed typical expression patterns in capillary endothelium in both sides of the spinal cord. In merged Beclin-1/LC3B images, double immunostaining demonstrates overlap between these autophagic biomarkers (orange, arrowheads) (a″, b″). At 7 days after tMCAO, extensive Beclin-1 (green, arrowheads) (c, d) and LC3B (red, arrowheads) (c′, d′) staining is shown in capillaries in the right and left sides of the spinal cord. Some capillaries showed lack of Beclin-1/LC3B overlay immunostaining; Beclin-1 protein immunostaining (green, asterisks) is clearly seen in merged images on both sides of the cord (c″, d″). Similarly, greater Beclin-1 (green, arrowheads) (e, f) and (e′, f′) LC3B (red, arrowheads) immunostaining is seen in the right and the left side capillaries 30 days after tMCAO versus controls. In merged images, omitted overlap between Beclin-1 and LC3B immunoexpressions (green, asterisks) was determined in the right (e″) and the left (f″) side capillaries. Nuclei are stained with DAPI in the merged images (a″–f″). Scale bar in a–f″ is 50 µm. (B) Quantitative analysis of Beclin-1 and LC3B immunostaining in capillary endothelium in control rats showed similar patterns on each side. Increases in Beclin-1 immunostaining on the right and left side spinal cord capillaries at 7 and 30 days post-tMCAO was noted versus that in controls (p < 0.001). Higher Beclin-1 immunostaining was noted in capillaries on the left versus the right side at 30 days post-tMCAO (p < 0.05). Nevertheless, there was upregulation of LC3B expression only at 7 days post-tMCAO in capillaries on the right (p < 0.05) and left (p < 0.01) sides versus controls. Higher LC3B expression was detected on the left side of ventral horn at 7 days versus 30 days post-tMCAO (p < 0.05). *p < 0.05; **p < 0.01; ***p < 0.001.