Journal: Journal of Histochemistry and Cytochemistry

Article Title: Morphological Characteristics of Neuronal Death After Experimental Subarachnoid Hemorrhage in Mice Using Double Immunoenzymatic Technique

doi: 10.1369/0022155419878181

Figure Lengend Snippet: Neuronal deoxyribonucleic acid (DNA) damage in the left temporal base cortex at 24 hr after subarachnoid hemorrhage (SAH) in mice. Serial sections of the same sample from a moderate-grade SAH mouse are used: (A) hematoxylin-eosin staining; (B) cresyl violet staining; (C) microtubule-associated protein 2 (MAP-2) staining; (D) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining counterstained with cresyl violet; (E) double immunolabeling with TUNEL and anti-MAP-2 antibody; (F and G) negative controls of double immunolabeling in which terminal deoxynucleotidyl transferase enzyme (F) or anti-MAP-2 antibody (G) is omitted, showing no crossreaction. Framed areas on panels A to E are magnified in the lower left insets to show morphologically apoptosis-appearing neurons in each staining: shrunken cytoplasm and dense nuclei profiled by hematoxylin and eosin (A), densely stained cells with cresyl violet (B), shrunken neuronal cell body revealed by MAP-2 (C), TUNEL-positive neuron having nucleoli clearly stained with cresyl violet (D), and TUNEL-positive neuron with MAP-2-positive cytoplasm (E). Arrows, morphologically apoptosis-appearing neurons containing small aggregates of chromatin; double arrows, corkscrew-like fibers of morphologically abnormal neurons; single arrowheads, small cells which may be glia; double arrowheads, small DNA-damaged cells densely stained with cresyl violet that may be glia or neuron; white arrows, morphologically intact neurons; white arrowheads, MAP-2-positive (F) or TUNEL-positive (G) neurons. Scale bar, 50 μm in panels and 10 μm in insets.

Article Snippet: Then, sections were used for immunostaining with anti-MAP-2 antibody, secondary antibody, and avidin–biotin complex and colored by alkaline phosphatase–mediated reaction with vector blue (cat. no. SK-5300; Vector).

Techniques: Staining, TUNEL Assay, Immunolabeling

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Morphological Characteristics of Neuronal Death After Experimental Subarachnoid Hemorrhage in Mice Using Double Immunoenzymatic Technique

doi: 10.1369/0022155419878181

Figure Lengend Snippet: Representative brain slice showing the left parietal (upper rectangle), lateral (middle rectangle), and temporal base (lower rectangle) cortices at 1.5 mm posterior to the bregma (A) and the number of apoptotic neurons in the left parietal, lateral, and temporal base cortex (B–D) at 24 hr after subarachnoid hemorrhage (SAH) in mice evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling and microtubule-associated protein 2 double immunolabeling. In the temporal base cortex, the moderate SAH group has significantly more deoxyribonucleic acid (DNA)-damaged neurons compared with the other groups (one-way ANOVA, *p<0.01). The moderate SAH group also has significantly more DNA-damaged neurons compared with the sham group in the lateral cortex (one-way ANOVA, **p<0.05). Scale bar, 1 mm.

Article Snippet: Then, sections were used for immunostaining with anti-MAP-2 antibody, secondary antibody, and avidin–biotin complex and colored by alkaline phosphatase–mediated reaction with vector blue (cat. no. SK-5300; Vector).

Techniques: Slice Preparation, TUNEL Assay, Immunolabeling

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Morphological Characteristics of Neuronal Death After Experimental Subarachnoid Hemorrhage in Mice Using Double Immunoenzymatic Technique

doi: 10.1369/0022155419878181

Figure Lengend Snippet: Representative terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and microtubule-associated protein 2 (MAP-2) double immunolabeling showing neuronal deoxyribonucleic acid (DNA) damage in the left parietal (A–C), lateral (D–F), and temporal base (G–I) cortices at 1.5 mm posterior to the bregma at 24 hr in sham (A, D and G), mild subarachnoid hemorrhage (SAH; B, E, and H), and moderate SAH (C, F, and I) mice. Note that little neuronal DNA damage is shown in the sham group, whereas the moderate SAH group extensively has DNA-damaged neurons. Framed areas on panels A to I are magnified in the lower left insets to show a representative neuron in each group: morphologically intact neurons with bright oval nuclei (A, D, and G), and DNA-damaged neurons with TUNEL-positive nuclei and shrunken MAP-2-positive cell body (B, C, E, F, H, and I). Arrows, DNA-damaged neurons; double arrows, corkscrew-like fibers of DNA-damaged neurons; white arrows, morphologically intact neurons. Scale bar, 50 μm in panels and 10 μm in insets.

Article Snippet: Then, sections were used for immunostaining with anti-MAP-2 antibody, secondary antibody, and avidin–biotin complex and colored by alkaline phosphatase–mediated reaction with vector blue (cat. no. SK-5300; Vector).

Techniques: TUNEL Assay, Immunolabeling

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Morphological Characteristics of Neuronal Death After Experimental Subarachnoid Hemorrhage in Mice Using Double Immunoenzymatic Technique

doi: 10.1369/0022155419878181

Figure Lengend Snippet: Representative brain slice showing the CA1 (upper rectangle) and CA3 (right rectangle) sectors of the left hippocampus at 1.5 mm posterior to the bregma (A), and representative terminal deoxynucleotidyl transferase dUTP nick end labeling and microtubule-associated protein 2 double immunolabeling showing neuronal deoxyribonucleic acid (DNA) damage in the CA1 (B–D) and CA3 (E–G) sectors of the left hippocampus at 24 hr in sham (B and E), mild subarachnoid hemorrhage (SAH; C and F), and moderate SAH (D and G) mice. A few DNA-damaged neurons (arrows and framed areas on panels C, D and G) are shown in both sectors in mild and moderate SAH mice. Framed areas on panels B to G are magnified in the lower left insets to show a representative neuron in each group: morphologically intact neurons with bright oval nuclei (B, E, and F), and DNA-damaged neurons (C, D, and G). Scale bar, 500 μm in panel A, 50 μm in panels B to G, and 10 μm in insets. Contrast and brightness enhancement on entire images are performed (A–G).

Article Snippet: Then, sections were used for immunostaining with anti-MAP-2 antibody, secondary antibody, and avidin–biotin complex and colored by alkaline phosphatase–mediated reaction with vector blue (cat. no. SK-5300; Vector).

Techniques: Slice Preparation, TUNEL Assay, Immunolabeling

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Morphological Characteristics of Neuronal Death After Experimental Subarachnoid Hemorrhage in Mice Using Double Immunoenzymatic Technique

doi: 10.1369/0022155419878181

Figure Lengend Snippet: Representative terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and microtubule-associated protein 2 (MAP-2) double immunolabeling showing neurons with different stages of deoxyribonucleic acid (DNA) damage in the left temporal base (A) and lateral (B) cortices at 24 hr in moderate-grade subarachnoid hemorrhage (SAH) mice. Arrows, relatively early stages of DNA-damaged neurons with TUNEL-positive shrunken nuclei and MAP-2-positive morphologically abnormal cell body; dotted arrows, late stages of DNA-damaged neurons in which MAP-2 immunoreactivity is shown only in shrunken cytoplasm; double arrows, vertically running curl-like structures of early stages of DNA-damaged neurons; double dotted arrows, TUNEL-positive but MAP-2-negative cells in empty holes, suggesting end stages of DNA-damaged neurons, glia, or endothelial cells; white arrows, morphologically intact neurons. Scale bar, 50 μm.

Article Snippet: Then, sections were used for immunostaining with anti-MAP-2 antibody, secondary antibody, and avidin–biotin complex and colored by alkaline phosphatase–mediated reaction with vector blue (cat. no. SK-5300; Vector).

Techniques: TUNEL Assay, Immunolabeling