Journal: Oncology Letters

Article Title: Synchronous liver metastasis at initial diagnosis of adrenal pheochromocytoma by CT: A case report

doi: 10.3892/ol.2024.14462

Figure Lengend Snippet: Postoperative pathological analysis. (A) HE staining results of adrenal mass. (B) HE staining results of liver mass. (C-E) Immunohistochemistry of liver mass: (C) Chromogranin A (+) and synaptophysin (+); (D) α-fetoprotein (−); and (E) cytokeratin (−) (magnification, ×400). Blue staining indicates negative expression, whereas brown staining indicates positive expression.

Article Snippet: The antibodies against chromogranin A (cat. no. #ZM-0076; dilution 1:200), synaptophysin (cat. no. #ZM-0246; dilution 1:200), CK7 (cat. no. #ZA-0573; dilution 1:200), CK20 (cat. no. #ZA-0574; dilution 1:200), α-fetoprotein (cat. no. #ZM-0009; dilution 1:200), and α-inhibin (cat. no. #ZM-0460; dilution 1:200) were from Origene Technologies, Inc.

Techniques: Staining, Immunohistochemistry, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Role of membrane sphingomyelin and ceramide in platform formation for Fas-mediated apoptosis

doi: 10.1084/jem.20041685

Figure Lengend Snippet: Characterization of WR/Fas-SM( − ) and WR/Fas-SMS1 cells. (A) SM synthase activity of WR/Fas-SM(−) (lane 1) and WR/Fas-SMS1 (lane 2) cells. The cellular lipids were labeled with [ 14 C]serine, extracted by the Bligh and Dyer method (reference ), and assessed by TLC. PE, phosphatidylethanolamine; PS, phosphatidylserine. (B) Analysis of membrane SM expression by confocal microscopy. Cells were stained with lysenin-MBP, and FITC-conjugated anti–mouse IgG mAb, then examined by laser scan confocal microscopy. (C) FACS analysis of membrane sphingolipids. To detect membrane SM, cells were stained with lysenin-MBP (Lysenin), CH11, and FITC-conjugated anti–mouse IgG mAb. Surface expression of ganglioside GM1, cholesterol, and human Fas were analyzed using FITC-conjugated CTx, cholesterol-PEG (cholesterol), or anti-Fas mAB (h-Fas), respectively.

Article Snippet: Aggregated Fas was detected by 3D5, a mouse mAb (IgG1) specific for the intracellular death domain of human Fas (Qbiogene).

Techniques: Activity Assay, Labeling, Expressing, Confocal Microscopy, Staining

Journal: The Journal of Neuroscience

Article Title: Pituitary Adenylate Cyclase-Activating Polypeptide Modulates Dendritic Spine Maturation and Morphogenesis via MicroRNA-132 Upregulation

doi: 10.1523/JNEUROSCI.2468-18.2019

Figure Lengend Snippet: PACAP significantly increased the number of function synapse in the primary cultured hippocampal neurons. A, Representative fluorescence images of the primary hippocampal neuronal dendrites at DIV21. The cells were fixed at DIV21 and immunostained using anti-synaptophysin (magenta) and anti-PSD95 (green) antibodies. Magnification of the area marked with a white dot box. Scale bar, 10 μm. B, Primary hippocampal neurons were treated for 2 d with 1, 10, or 100 nm PACAP. Scale bar, 10 μm. C, Quantification of the number of PSD-95-positive spines that were apposed to synaptophysin-positive presynaptic terminals. Values indicate mean ± SEM on 60 neurons from three independent experiments. **p < 0.01 versus vehicle (one-way ANOVA followed by Tukey–Kramer test). D, Immunoblot of cell surface biotinylated α1 sodium potassium ATPase, NR1, and NR2B from the primary cultured hippocampal neurons at DIV14. Surface biotinylation assay was performed after 2 d treatment with vehicle or 10 nm PACAP. E, Quantification of the cell surface expression levels of NR1 and NR2B were normalized to the levels of α1 sodium potassium ATPase. Values indicate mean ± SEM from five independent experiments. *p < 0.05 versus vehicle (Student's t test). F, Representative immunoblots showing expression of pERK1/2 and ERK2. Synaptic NMDAR activity in the primary cultured hippocampal neurons at DIV14 were treated with vehicle or 500 μm 4-AP and 50 μm bicuculline (bic) for 5 or 30 min after 2 d treatment with vehicle or 10 nm PACAP. 4-AP is a potassium channel blocker, and bicuculline is a GABAA receptor blocker. G, Quantification of the expression levels of pERK2 were normalized to the levels of ERK2. Values indicate mean ± SEM from four independent experiments. *p < 0.05 versus vehicle or 0 min (two-way ANOVA followed by Tukey–Kramer test).

Article Snippet: For predendritic and postdendritic spine labeling, the primary antibodies were used a rabbit polyclonal anti-PSD-95 antibody (1:500; catalog #3450S, RRID: AB_2292883 , Cell Signaling Technology) and a mouse monoclonal anti-synaptophysin antibody (1:2000; catalog #611880, RRID: AB_399360 , BD Biosciences) diluted in 0.2% Triton X-100 in PBS supplemented with 3% BSA.

Techniques: Cell Culture, Fluorescence, Western Blot, Surface Biotinylation Assay, Expressing, Activity Assay

Journal: The Journal of Neuroscience

Article Title: Pituitary Adenylate Cyclase-Activating Polypeptide Modulates Dendritic Spine Maturation and Morphogenesis via MicroRNA-132 Upregulation

doi: 10.1523/JNEUROSCI.2468-18.2019

Figure Lengend Snippet: Viral expression of miR-132 increased dendritic spine density in the primary hippocampal neurons from PACAP−/− mice. A, Representative fluorescence images of the primary hippocampal neuronal dendrites at DIV21. The cells were fixed at DIV21 and immunostained using anti-synaptophysin (magenta) and anti-PSD95 (green) antibodies. Scale bar, 10 μm. B, Quantification of the number of PSD-95-positive spines that were apposed to synaptophysin-positive presynaptic terminals. Values indicate mean ± SEM on 80 neurons from four independent experiments. *p < 0.05 versus vehicle (one-way ANOVA followed by Tukey–Kramer test). C, Expression levels of miR-132 in WT and PACAP−/− mice primary hippocampal neurons were analyzed by qRT-PCR. Primary hippocampal neurons were harvested at DIV20. Values indicate mean ± SEM from nine independent experiments. *p < 0.05 versus WT (one-way ANOVA followed by Tukey-kramer test). D, Representative fluorescence images of primary hippocampal neuron dendrites at DIV21. Primary hippocampal neurons were transduced with miR-132 Venus or control-Venus lentivirus at DIV14 and treated with 10 nm PACAP at DIV19. The cells were fixed at DIV21 and immunostained with anti-GFP (green) and anti-PSD95 (magenta) antibodies at DIV21. Scale bar, 10 μm. E–G, Quantification of the number of total spines (E), PSD-95-positive spines (F), and -negative spines (G). Values indicate mean ± SEM of 60 neurons from three independent experiments. **p < 0.01 versus Venus or WT (two-way ANOVA followed by Tukey–Kramer test).

Article Snippet: For predendritic and postdendritic spine labeling, the primary antibodies were used a rabbit polyclonal anti-PSD-95 antibody (1:500; catalog #3450S, RRID: AB_2292883 , Cell Signaling Technology) and a mouse monoclonal anti-synaptophysin antibody (1:2000; catalog #611880, RRID: AB_399360 , BD Biosciences) diluted in 0.2% Triton X-100 in PBS supplemented with 3% BSA.

Techniques: Expressing, Fluorescence, Quantitative RT-PCR, Transduction

Journal: Neurobiology of Aging

Article Title: Alterations in the nigrostriatal system following conditional inactivation of α-synuclein in neurons of adult and aging mice

doi: 10.1016/j.neurobiolaging.2020.02.026

Figure Lengend Snippet: Expression of presynaptic proteins in the dorsal striatum of 18-month-old Snca flox/- and Snca flox/+ mice injected with tamoxifen and Snca flox/- mice injected with vehicle at the age of 6 months. (A) The upper panel shows a representative Western blot image illustrating the expression of tyrosine hydroxylase (TH) and synaptophysin with β-actin as a loading control. Individual mouse striata were analyzed. The Cy5-conjugated secondary antibody was used for the detection of these proteins. A bottom part of the same membrane was probed with an antibody against mouse α-synuclein (middle panel), and then, without stripping, was re-probed with an antibody against β-synuclein (bottom panel). HRP- conjugated secondary antibody and chemiluminescent detection were used for the detection of both synucleins. For quantification, protein bands were scanned, levels of TH (B) and synaptophysin (C) were normalized to levels of β-actin in studied striatal samples and then to the average level in control Snca flox/+ mice. Bar charts show means±SEM of normalized values obtained by Western blot analysis of 6 vehicle-injected (veh) and 6 tamoxifen-injected (TMX) Snca flox/- mice and 4 tamoxifen-injected Snca flox/+ (TMX (α + )) mice. Statistical analysis using the Kruskal-Wallis test revealed no statistically significant difference between groups ( p = 0.9704; Kruskal-Wallis statistic 0.06548 for TH and p = 0.2438; Kruskal-Wallis statistic 2.890 for synaptophysin).

Article Snippet: Primary antibodies against α-synuclein (mouse monoclonal antibody, clone 42, BD Transduction Laboratories, diluted 1:1000), β-synuclein (rabbit monoclonal, clone EP1537Y, Abcam, diluted 1:5000), TH (mouse monoclonal, clone TH-2, Sigma, diluted 1:5000), synaptophysin (mouse monoclonal, clone 2, BD Transduction Laboratories, diluted 1:10,000), aldehyde dehydrogenase ALDH1a1 (rabbit polyclonal ABD12, Merck, diluted 1:4000) and β-actin (mouse monoclonal, clone AC-15, Sigma, diluted 1:10,000) were used.

Techniques: Expressing, Injection, Western Blot, Stripping Membranes

Journal: Neurobiology of Aging

Article Title: Alterations in the nigrostriatal system following conditional inactivation of α-synuclein in neurons of adult and aging mice

doi: 10.1016/j.neurobiolaging.2020.02.026

Figure Lengend Snippet: Expression of presynaptic proteins in the dorsal striatum of 18-month-old Snca flox/- mice injected with tamoxifen or vehicle at the age of 12 months. (A) The upper panel shows a representative Western blot image illustrating the expression of tyrosine hydroxylase (TH) and synaptophysin with β-actin as a loading control. Individual mouse striata were analyzed. The Cy5-conjugated secondary antibody was used for the detection of these proteins. A bottom part of the same membrane was probed with an antibody against mouse α-synuclein and HRP- conjugated secondary antibody following chemiluminescent detection. For quantification, protein bands were scanned, levels of TH (B) and synaptophysin (C) in each striatal sample were normalized to the level of β-actin in this sample and then to the average level in control vehicle-injected mice. Bar charts show means±SEM of normalized values obtained by Western blot analysis of 6 vehicle-injected (veh) and 8 tamoxifen-injected (TMX) Snca flox/- mice. Statistical analysis using the Mann-Whitney test revealed no statistically significant difference between groups ( p = 0.5080; sum of ranks (137, 163); Mann-Whitney U = 58 for TH and p = 0.2284; sum of ranks (55, 50); Mann-Whitney U = 14 for synaptophysin).

Article Snippet: Primary antibodies against α-synuclein (mouse monoclonal antibody, clone 42, BD Transduction Laboratories, diluted 1:1000), β-synuclein (rabbit monoclonal, clone EP1537Y, Abcam, diluted 1:5000), TH (mouse monoclonal, clone TH-2, Sigma, diluted 1:5000), synaptophysin (mouse monoclonal, clone 2, BD Transduction Laboratories, diluted 1:10,000), aldehyde dehydrogenase ALDH1a1 (rabbit polyclonal ABD12, Merck, diluted 1:4000) and β-actin (mouse monoclonal, clone AC-15, Sigma, diluted 1:10,000) were used.

Techniques: Expressing, Injection, Western Blot, MANN-WHITNEY

Journal: Acta Neuropathologica Communications

Article Title: Super-resolution microscopy reveals γ-secretase at both sides of the neuronal synapse

doi: 10.1186/s40478-016-0296-5

Figure Lengend Snippet: STORM imaging of γ-secretase in synapses. ( a ) Localization of active γ-secretase at the presynaptic zone showing γ-secretase ( red ) and the presynaptic marker synaptophysin ( blue ). The upper left panel shows a typical STORM image on a brightfield overlay to the left, with increased zooming in the middle and right panels , displaying an individual synaptic region to the right. The lower panel shows the same synapse displayed in 3D view from three different angles where red , green and blue lines represent X, Y and Z directions, respectively. ( b ) Localization of active γ-secretase at the postsynaptic zone showing γ-secretase ( red ) and the postsynaptic marker PSD95 ( blue ). The upper left panel shows a typical STORM image on a brightfield overlay to the left, with increased zooming in the middle and right panels . The lower panel shows the same synapse displayed in 3D view from three different angles where red , green and blue lines in the symbol represent X, Y and Z directions, respectively

Article Snippet: For presynaptic triple staining; the cells were incubated with 200 nM GTB and mouse monoclonal synaptophysin antibody diluted 1:200 (ADI-VAM-SV011-F, Enzo) in 3 % NGS at 4 °C ON.

Techniques: Imaging, Marker

Journal: Acta Neuropathologica Communications

Article Title: Super-resolution microscopy reveals γ-secretase at both sides of the neuronal synapse

doi: 10.1186/s40478-016-0296-5

Figure Lengend Snippet: STED imaging of γ-secretase in synapses with triple-staining. γ-Secretase and the synaptic markers were visualized to show the synaptic location of γ-secretase together with f-actin to outline the cellular structure. ( a ) Localization of γ-secretase ( red ) at the synapse using synaptophysin ( blue ) as a presynaptic marker. Dendritic spine structure was visualized with Phalloidin as a marker for f-actin ( white ). The distribution of γ-secretase throughout the synaptophysin-stained region is marked by the upper arrow , whereas the γ-secretase staining in the dendritic spine is shown by the lower arrow . ( b ) Localization of γ-secretase ( red ) at the synapse using PSD95 ( green ) as a postsynaptic marker and phalloidin as a marker for f-actin ( white ). The presence of γ-secretase in the postsynaptic compartment is shown, where the postsynaptic marker PSD95 is found in dense regions inside the dendritic spine head ( arrow )

Article Snippet: For presynaptic triple staining; the cells were incubated with 200 nM GTB and mouse monoclonal synaptophysin antibody diluted 1:200 (ADI-VAM-SV011-F, Enzo) in 3 % NGS at 4 °C ON.

Techniques: Imaging, Staining, Marker

Journal: Acta Neuropathologica Communications

Article Title: Super-resolution microscopy reveals γ-secretase at both sides of the neuronal synapse

doi: 10.1186/s40478-016-0296-5

Figure Lengend Snippet: Localization of γ-secretase at the postsynaptic plasma membrane. ( a ) STED images showing localization of γ-secretase (red) very close to the synaptic cleft in the dendritic spines ( white arrows ). Synaptophysin (blue) and PSD95 (green) mark the pre- and postsynaptic sides, respectively. The close-ups are taken from Additional file : Figure S5b (a1) and S5d (a2). ( b ) STORM imaging of γ-secretase ( red ) and NMDAR2B ( green ). The image shows a typical STORM image on a brightfield overlay ( left ), with increased magnification in the middle and right panels , displaying a close-up from one individual postsynaptic membrane region to the right. ( c ) The same postsynaptic membrane region as the right image in (b) is displayed in 3D view from three different angles where red , green and blue lines represent X, Y and Z directions, respectively

Article Snippet: For presynaptic triple staining; the cells were incubated with 200 nM GTB and mouse monoclonal synaptophysin antibody diluted 1:200 (ADI-VAM-SV011-F, Enzo) in 3 % NGS at 4 °C ON.

Techniques: Clinical Proteomics, Membrane, Imaging