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

Journal: Frontiers in Immunology

Article Title: Staphylococcus epidermidis Sensitizes Perinatal Hypoxic-Ischemic Brain Injury in Male but Not Female Mice

doi: 10.3389/fimmu.2020.00516

Figure Lengend Snippet: S. epidermidis 24 h prior to hypoxia-ischemia potentiates gray matter injury in neonatal male mice. PND4 mice were injected with saline or S. epidermidis and subjected to hypoxia-ischemia 24 h later. (A) Gray matter brain injury was assessed by microtubule-associated protein 2 (MAP-2) immunohistochemistry at PND14 in the entire cerebral hemisphere, cerebral cortex, hippocampus, thalamus, and striatum ( n = 12 saline males; n = 11 S. epidermidis males; n = 17 saline females, and n = 13 S. epidermidis females). (B) Representative images (4 × objective lens) of PND14 male mice brain sections stained with MAP-2 at the hippocampal and striatal levels following saline or S. epidermidis injection at PND4 in combination with hypoxia-ischemia 24 h later. Images of CA3 region of the hippocampus and striatum captured on a light microscope with a 20× objective lens. Data are presented as median and 10–90 th percentile. Statistical comparison between the S. epidermidis and saline groups for each brain region was performed using Two-way ANOVA with Sidak's multiple comparison post-hoc test; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Sections were incubated at 4°C overnight with primary antibody against microtubule-associated protein-2 (MAP-2; clone HM-2, 1:1,000; Sigma-Aldrich catalog # M4403) or myelin basic protein (MBP; clone SMI-94, 1:1,000; BioLegend catalog # 836504), followed by 1 h of incubation with horse-anti-mouse biotinylated secondary antibody (1:250; Vector Laboratories catalog # BA-2001) and VECTASTAIN Elite ABC HRP Kit (Vector Laboratories) according to manufacturer's instructions.

Techniques: Injection, Immunohistochemistry, Staining, Light Microscopy