Journal: Journal of neuropathology and experimental neurology

Article Title: Synaptophysin Is a Reliable Marker for Axonal Damage.

doi: 10.1093/jnen/nlw114

Figure Lengend Snippet: FIGURE 1. (A–C) Representative sections illustrate immunohistochemical staining for synaptophysin (Syp) in the cerebral cortex and hippocampus (A), cerebellum (B), and corpus callosum (C) of untreated control mice. (D) Accumulation of synaptophysin- positive vesicles ¼ spheroids/ovoids/bulbs in the corpus callosum of a cuprizone-treated mouse. Arrows indicate the different sizes of synaptophysin-positive-spheroids; small [S],<2 mm (as in panel C); middle-sized [M], 2–7.5 mm; large [L], 8–15 mm, pathological size. (E, F) Graphs show numbers of different sized synaptophysin-positive spheroids in the corpus callosum during acute (5 weeks, E) and chronic (12 weeks, F) demyelination followed by remyelination after stopping cuprizone. Significant effects between different time points were calculated separately for each type/size of bulbs (*p <0.05, **p < 0.01, **p < 0.001). Significant effects for different cuprizone time treatments in comparison to the untreated control: #p< 0.05, ##p < 0.01, ###p < 0.001), n ¼ 4–6. (G, H) Representative images show synaptophysin-positive bulbs in the demyelinated hippocampus (G) and internal capsule (H). (I-K) Representative images show that synaptophysin does not colocalize with the oligodendroglial marker Nogo-A (I), the astrocytic marker GFAP (J), or with the microglial marker Iba-1 (K). Scale bar in (K)¼ 100 mm, applies to (I) and (J).

Article Snippet: We used 2 different antibodies for synaptophysin: a mouse monoclonal IgG from Serotec and rabbit monoclonal from Abcam.

Techniques: Immunohistochemical staining, Staining, Control, Comparison, Marker

Journal: Journal of neuropathology and experimental neurology

Article Title: Synaptophysin Is a Reliable Marker for Axonal Damage.

doi: 10.1093/jnen/nlw114

Figure Lengend Snippet: FIGURE 2. (A, B) Graphs represent the quantitative analysis of APP, synaptophysin (Syp), and double-positive spheroids in the corpus callosum of cuprizone-treated mice with acute demyelination (A) and chronic demyelination (B). Two-way ANOVA analysis identify that there were no significant differences between numbers of APP- and synaptophysin-positive spheroids during

Article Snippet: We used 2 different antibodies for synaptophysin: a mouse monoclonal IgG from Serotec and rabbit monoclonal from Abcam.

Techniques:

Journal: Journal of neuropathology and experimental neurology

Article Title: Synaptophysin Is a Reliable Marker for Axonal Damage.

doi: 10.1093/jnen/nlw114

Figure Lengend Snippet: FIGURE 3. Immunohistochemical staining of the corpus callosum evaluating phosphorylation of neurofilaments and their colocalization with synaptophysin (Syp) during cuprizone-induced demyelination. (A–H) SMI-32 detecting non-phosphorylated heavy neurofilaments are shown in panels (A, C, E, G). SMI-312 detecting phosphorylated neurofilaments H and M are

Article Snippet: We used 2 different antibodies for synaptophysin: a mouse monoclonal IgG from Serotec and rabbit monoclonal from Abcam.

Techniques: Immunohistochemical staining, Staining, Phospho-proteomics

Journal: Journal of neuropathology and experimental neurology

Article Title: Synaptophysin Is a Reliable Marker for Axonal Damage.

doi: 10.1093/jnen/nlw114

Figure Lengend Snippet: FIGURE 5. (A-P) Representative sections show immunostaining in the corpus callosum for synaptophysin/Iba-1 (A-F) and synaptophysin/ PLP (G–L) during acute and chronic cuprizone induced demyelination and subsequent remyelination. Inserts in (A–F) show the morphology/shape of microglia identifying their activation state. Inserts in (G–L) show higher magnification of synaptophysin accumulations. Panels (M) and (N) are confocal images from RCA-1 (microglia activation marker, green) and synaptophysin (red) during inflammation in cuprizone-treated mice. Panels (O) and (P) show remyelinated axons (PLP, green) with internal synaptophysin accumulation (red).

Article Snippet: We used 2 different antibodies for synaptophysin: a mouse monoclonal IgG from Serotec and rabbit monoclonal from Abcam.

Techniques: Immunostaining, Activation Assay, Marker

Journal: Journal of neuropathology and experimental neurology

Article Title: Synaptophysin Is a Reliable Marker for Axonal Damage.

doi: 10.1093/jnen/nlw114

Figure Lengend Snippet: FIGURE 6. The appearance of synaptophysin (Syp)-positive pathological (medium-and large-sized) spheroids was compared and correlated with the extent of demyelination (proteolipid protein: PLP, myelin basic protein: MBP) and microglia infiltration (Iba-1) induced by cuprizone feeding (acute or chronic). (A, B) Graphs show the extent of microglia accumulation, appearance of pathological synaptophysin bulbs and the extent of de- and remyelination as judged by scoring of MBP and PLP immunostaining. Score of 3 represents complete myelination; score of 0 represents complete demyelination. (C–H) Correlation analyses of synaptophysin-positive bulbs with the extent of de- and remyelination and microglia numbers (n ¼ 5–6).

Article Snippet: We used 2 different antibodies for synaptophysin: a mouse monoclonal IgG from Serotec and rabbit monoclonal from Abcam.

Techniques: Immunostaining

Journal: Journal of neuropathology and experimental neurology

Article Title: Synaptophysin Is a Reliable Marker for Axonal Damage.

doi: 10.1093/jnen/nlw114

Figure Lengend Snippet: FIGURE 8. (A–C) Graphs show microglia/macrophage infiltration (A) and the course of demyelination (B) during Theiler’s virus induced encephalomyelitis in the thoracic segment of murine spinal cord. (C) Panel shows the appearance of pathologic accumulation of synaptophysin (Syp). Significant effects between different time points were indicated as: *p < 0.05, **p < 0.01, **p < 0.001. Significant effects for different time treatments in comparison to the untreated control are indicated as: # p < 0.05, ## p < 0.01, ### p < 0.001), n ¼ 4–6. (D–H) Representative images of thoracic spinal cord segment illustrate infiltration of microglia/macrophage at different days after the injection of Theiler’s murine encephalomyelitis virus. (I–U) Panel (I) shows the appearance of synaptophysin-positive bulbs exclusively in the demyelinated area; (J) and (K) illustrate the appearance of synaptophysin-positive spheroids surrounded by activated microglia/macrophages. (L–N) Colocalization of synaptophysin and APP in the spheroid structures. (O–U) Serial staining of the same spinal cord area during Theiler’s virus induced encephalomyelitis (O, synaptophysin/PLP double staining; P, synaptophysin/Iba-1 double staining; R, synaptophysin/APP double staining; S–U, higher magnification of area shown in R).

Article Snippet: We used 2 different antibodies for synaptophysin: a mouse monoclonal IgG from Serotec and rabbit monoclonal from Abcam.

Techniques: Virus, Comparison, Control, Injection, Staining, Double Staining

Journal: Journal of neuropathology and experimental neurology

Article Title: Synaptophysin Is a Reliable Marker for Axonal Damage.

doi: 10.1093/jnen/nlw114

Figure Lengend Snippet: FIGURE 9. Representative images show the expression of synaptophysin (Syp) in CNS tissue of 2 different patients with MS. (A– H) Different immunohistochemical stainings from a chronic active MS lesion (serial sections). Panels (E) and (F) show the higher magnifications from boxed areas 1 in (C) and (D). Panels (G) and (H) demonstrate higher magnification from boxed areas 2 in (C) and D. (I–K) Representative double immunostaining for synaptophysin and Iba-1 demonstrate microglia accumulation (I) and proving colocalization of APP and synaptophysin (J, K). (L–O) Demyelination/inflammation/axonal damage in another MS lesion. DAB staining in (L) and (M) illustrate the extent of demyelination (PLP) and inflammation (LN3 marks HLA-DR-positive cells: lymphocytes and macrophages) in this MS lesion. Panels N and O show higher magnification of the boxed are from this lesion in (L) and (M) demonstrating double expression of APP and synaptophysin in (N) and double staining of PLP and synaptophysin in (O). (P–U) Corpora amylacea are immunostain-positive for APP (P, S), and NeuN (U), but not synaptophysin (R, T).

Article Snippet: We used 2 different antibodies for synaptophysin: a mouse monoclonal IgG from Serotec and rabbit monoclonal from Abcam.

Techniques: Expressing, Immunohistochemical staining, Double Immunostaining, Staining, Double Staining

Journal: Neural Regeneration Research

Article Title: Necroptosis plays a crucial role in the exacerbation of retinal injury after blunt ocular trauma

doi: 10.4103/1673-5374.353848

Figure Lengend Snippet: Damage to the ONL and INL worsens on the seventh day after blunt ocular trauma. (A) Representative images of ChAT staining (green; cholinergic cells). Compared with the number of ChAT-positive cells in the control group, the number of ChAT-positive cells began to decline on the seventh day after injury. (B) The proportion of ChAT-positive cells was recorded after blunt ocular trauma and normalized to data from the control group. (C) Representative images of calretinin staining (green; amacrine cells). Compared with the number of calretinin-positive cells in the control group, the number of calretinin-positive cells also began to decline on the seventh day after injury. (D) The proportion of calretinin-positive cells was recorded after blunt trauma and normalized to data from the control group. (E) Representative images of calbindin-D staining (green; horizontal cells). Compared with the number of calbindin-D-positive cells in the control group, the number of calbindin-D-positive cells did not change. (F) The proportion of calbindin-D-positive cells was recorded after blunt trauma and normalized to data from the control group. (G) Representative images of PKCα (PKC, red; bipolar cells) and synaptophysin (Syn, green; photoreceptor ribbon synapses) staining. Compared with the number of PKC-positive cells in the control group, the number of PKC-positive cells significantly decreased on the first day after injury. Compared with the number of PKCα and synaptophysin double-positive cells in the control group, the number of PKCα and synaptophysin double-positive cells was reduced on the seventh day after injury. (H) The proportion of PKC-positive cells was recorded after blunt ocular trauma and normalized to data from the control group. (I) The percentage overlap of PKCα and synaptophysin double-positive cells was recorded after blunt ocular trauma. Scale bar: 100 μm. Data are shown as the mean ± SEM ( n = 5 per group). * P < 0.05, vs . control group (Student’s t -test). ChAT: Choline acetyltransferase; PKC: protein kinase C.

Article Snippet: Sections were then incubated at 4°C overnight with one or more of the following primary antibodies [all diluted 1:100 by a fresh blocking buffer (0.01 M PBS containing 0.3% Triton X-100 and 3% bovine serum albumin)]: synaptophysin (Syn; rabbit; Aviva Systems Biology, San Diego, CA, USA; Cat# ARP45435, RRID: AB_2048301), Ki67 (rabbit; Proteintech, Wuhan, China; Cat# 27309-1-AP, RRID: AB_2756525), glutamine synthetase (GS; mouse; Proteintech; Cat# 66323-2-Ig, RRID: AB_2881704), CD45 (rabbit; Proteintech; Cat# 20103-1-AP, RRID: AB_2716813), sex determining region Y-box 2 (SOX2) (rabbit; Novus, Littleton, CO, USA; Cat# NB110-37235, RRID: AB_792070), protein kinase C (PKC; mouse; Enzo, New York, NY, USA; Cat# ADI-KAM-PK020, RRID: AB_10998587), inducible nitric oxide synthase 2 (iNOS) (mouse; Santa Cruz Biotechnology, Dallas, TX, USA; Cat# sc-7271, RRID: AB_2891105), glial fibrillary acidic protein (GFAP; mouse; Millipore, Burlington, MA, USA; Cat# MAB3402, RRID: AB_94844), calretinin (rabbit; Abcam, Cambridge, UK; Cat# ab702, RRID: AB_305702), choline acetyltransferase (ChAT; rabbit; Abcam; Cat# ab6168, RRID: AB_2244866), ionized calcium binding adapter molecule 1 (Iba1) (rabbit; Abcam; Cat# ab178847, RRID: AB_2832244), Iba1 (mouse; Abcam; Cat# ab15690, RRID: AB_2224403), calbindin-D (rabbit; Sigma, St. Louis, MO, USA; Cat# c2724, RRID: AB_258818), or CD16/32 (rabbit; Abcam; Cat# ab223200).

Techniques: Staining, Control