Journal: BMC Musculoskeletal Disorders

Article Title: Decreased Semaphorin3A expression correlates with disease activity and histological features of rheumatoid arthritis

doi: 10.1186/1471-2474-14-40

Figure Lengend Snippet: Histological/immunohistochemical analysis of Sema3A, NRP1, VEGF 165 and CD3 in OA and RA synovial tissue. Representative HE staining of OA ( A ) and RA ( D ) synovial tissues. OA synovial tissues contain lining (LL) and sublining (SL) layers. RA synovial tissues are marked by the hyperplasia of synovial tissues in the lining layer and numerous infiltrated inflammatory cells (IC) in the sublining layer. Sema3A expression was detected in the lining layer and inflammatory cells in the sublining layer of OA ( B ) and RA ( E ) synovial tissues. The density of the Sema3A signal in the lining layer was lower in RA than OA. Peptide-neutralized anti-Sema3A antibodies did not stain tissue sections from OA ( C ) and RA ( F ). NRP1 and VEGF 165 localized to the same areas as Sema3A in OA. VEGF 165 expression in the lining layer in RA tissues was similar with OA ( G, J ). The NRP1 expression level in the lining layer of RA was similar to OA ( H, K ). T cells (CD3) and B cells (CD20) were detected among inflammatory cells in the sublining layer of OA and RA synovial tissues ( I, L, M, N ). The numbers of T cells and B cells were higher in RA synovial tissues compared with OA. Sections were counterstained with hematoxylin. Scale bars = 50 μm in the whole image view and 25 μm in the magnified view. Immunostaining of lining layer Sema3A was significantly decreased in RA (n = 12) synovial tissues compared with OA (n = 12) subjects ( O ). Results are presented as relative values compared with OA subjects. The box plots demonstrate the 10th and 90th percentile (whiskers), the 25th and 75th percentile, and the median. P values were obtained using the Mann–Whitney U -test.

Article Snippet: The assay was performed in triplicate in optical 96-well reaction plates covered with optical adhesive cover in a volume of 10 μl containing 0.5 μl Taqman Gene Expression Assay 20X for human Sema3A (assay ID Hs01085496_m1, GenBank accession number NM_006080, Applied Biosystems LLC), VEGF-A (assay ID Hs00173626_m1, GenBank accession number NG_008732, Applied Biosystems LLC), NRP1 (assay ID Hs00826128_m1, GenBank accession number NM_003873; Applied Biosystems LLC) and β-actin (assay ID 4326315E, GenBank accession number NM_001101; Applied Biosystems LLC), 5 μl Taqman Fast Advanced Master Mix 2X, 2 μl cDNA template and 2.5 μl RNase-free water.

Techniques: Immunohistochemical staining, Staining, Expressing, Immunostaining, MANN-WHITNEY

Journal: BMC Musculoskeletal Disorders

Article Title: Decreased Semaphorin3A expression correlates with disease activity and histological features of rheumatoid arthritis

doi: 10.1186/1471-2474-14-40

Figure Lengend Snippet: Expression of Sema3A, VEGF-A and NRP1 mRNA in synovial tissues. Expression levels of Sema3A , VEGF-A and NRP1 mRNA were measured using real-time PCR. The mRNA levels were normalized to the expression of β-actin . The box plots demonstrate the 10th and 90th percentile (whiskers), the 25th and 75th percentile, and the median. Sema3A mRNA levels were significantly decreased in RA (n = 30) synovial tissues compared with OA (n = 23) ( A ). VEGF-A and NRP1 mRNA levels were not significantly altered ( B, C ). Correlation of Sema3A mRNA levels and DAS28-CRP ( D ); VEGF-A mRNA levels and DAS28-CRP ( E ); and Sema3A / VEGF-A mRNA levels and DAS28-CRP ( F ). P values were obtained using the Mann–Whitney U -test. were examined using Spearman’s rank correlation coefficient.

Article Snippet: The assay was performed in triplicate in optical 96-well reaction plates covered with optical adhesive cover in a volume of 10 μl containing 0.5 μl Taqman Gene Expression Assay 20X for human Sema3A (assay ID Hs01085496_m1, GenBank accession number NM_006080, Applied Biosystems LLC), VEGF-A (assay ID Hs00173626_m1, GenBank accession number NG_008732, Applied Biosystems LLC), NRP1 (assay ID Hs00826128_m1, GenBank accession number NM_003873; Applied Biosystems LLC) and β-actin (assay ID 4326315E, GenBank accession number NM_001101; Applied Biosystems LLC), 5 μl Taqman Fast Advanced Master Mix 2X, 2 μl cDNA template and 2.5 μl RNase-free water.

Techniques: Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: BMC Musculoskeletal Disorders

Article Title: Decreased Semaphorin3A expression correlates with disease activity and histological features of rheumatoid arthritis

doi: 10.1186/1471-2474-14-40

Figure Lengend Snippet: Immunohistochemical and double immunofluorescence staining of lymphocyte aggregates. NRP1 was abundantly expressed by inflammatory cells of lymphocytes aggregates ( A ). Immunohistochemical staining of sequential sections showed that NRP1-positive cells in lymphocytes aggregates also expressed CD3 ( B ) or CD20 ( C ). Micrographs show the double immunofluorescence detection of NRP1 ( D , green) and CD20 ( E , red) with the merged images ( F ). The NRP1 positive signal colocalized with the CD20 positive signal in the inflammatory cells of lymphocyte aggregates. A–C, scale bars = 50 μm; D–F, scale bars = 10 μm.

Article Snippet: The assay was performed in triplicate in optical 96-well reaction plates covered with optical adhesive cover in a volume of 10 μl containing 0.5 μl Taqman Gene Expression Assay 20X for human Sema3A (assay ID Hs01085496_m1, GenBank accession number NM_006080, Applied Biosystems LLC), VEGF-A (assay ID Hs00173626_m1, GenBank accession number NG_008732, Applied Biosystems LLC), NRP1 (assay ID Hs00826128_m1, GenBank accession number NM_003873; Applied Biosystems LLC) and β-actin (assay ID 4326315E, GenBank accession number NM_001101; Applied Biosystems LLC), 5 μl Taqman Fast Advanced Master Mix 2X, 2 μl cDNA template and 2.5 μl RNase-free water.

Techniques: Immunohistochemical staining, Double Immunofluorescence Staining, Staining, Immunofluorescence

Journal: PLoS ONE

Article Title: Osx-Cre Targets Multiple Cell Types besides Osteoblast Lineage in Postnatal Mice

doi: 10.1371/journal.pone.0085161

Figure Lengend Snippet: (A–B) Double immunostaining for EGFP (A) and perilipin (B) on longitudinal sections of tibias from two-month-old Osx-Cre; R26-mT/mG mice. (C) Co-localization of EGFP and perilipin. Arrows denote co-expression of GFP and perilipin. Green: EGFP; magenta: perilipin; blue: DAPI.

Article Snippet: For detection of GFP, perilipin or αSMA, immunostaining was performed on cryostat sections using a chicken polyclonal GFP antibody (1∶2500; Abcam, Cambridge, MA), or rabbit monoclonal perilipin antibody (1∶100; Cell Signaling Technology, Danvers, MA), or mouse monoclonal αSMA antibody (1∶500, Sigma, St. Louis, MO).

Techniques: Double Immunostaining, Expressing

Journal: PLoS ONE

Article Title: Osx-Cre Targets Multiple Cell Types besides Osteoblast Lineage in Postnatal Mice

doi: 10.1371/journal.pone.0085161

Figure Lengend Snippet: (A–B) Images for direct fluorescence from tdTomato (A) or EGFP (B) in whole-mount gonadal fat depots from two-month-old Osx-Cre ; R26-mT/mG mice. (C–F) Direct fluorescence for tdTomato (C) and immunofluorescence for perilipin (D) and EGFP (E) on sections of gonadal fat depots from two-month-old Osx-Cre;R26-mT/mG mice. (G–K) Imaging of longitudinal sections of an intramuscular fat depot associated with a tibia from two-month-old Osx-Cre;R26-mT/mG mice. G: perilipin immunofluorescence; H: EGFP immunofluorescence; I: direct fluorescence for tdTomato; J: merged view of G–I; K: DAPI staining. Arrow: GFP-positive periosteum.

Article Snippet: For detection of GFP, perilipin or αSMA, immunostaining was performed on cryostat sections using a chicken polyclonal GFP antibody (1∶2500; Abcam, Cambridge, MA), or rabbit monoclonal perilipin antibody (1∶100; Cell Signaling Technology, Danvers, MA), or mouse monoclonal αSMA antibody (1∶500, Sigma, St. Louis, MO).

Techniques: Fluorescence, Immunofluorescence, Imaging, Staining

Journal: Oncotarget

Article Title: Menin mediates Tat-induced neuronal apoptosis in brain frontal cortex of SIV-infected macaques and in Tat-treated cells

doi: 10.18632/oncotarget.14993

Figure Lengend Snippet: Menin expression is visible in nuclei, with some cytoplasmic expression, in the frontal cortex of SIV-infected A . and control B . macaques. There are more menin-positive cells in SIV-infected macaques A, C . than in the controls B, D . IOD analysis shows more menin immunostaining in the frontal cortex of SIV-infected macaques ( #1-#9 ) compared with control macaques ( #10–13 ), integrated optical density = optical density - background E . data are expressed as mean ± SD, * P < 0.05. Western blotting also shows increased menin expression in SHIV -SF162.P4 -infected macaques ( #1 ) compared with control macaques ( #12 ). A specific β-actin band (about 43 kDa) is shown under the menin band (about 68 kDa) F . Original magnification: (A-B) 200×; (C, D) 400×. (A, C) from macaque #9 , (B, D) from #10 . Arrows showing positive cells of IHC staining.

Article Snippet: We also performed double-labeled IHC against menin (1:200, rabbit, Cell Signaling Technology, MA, USA, #6891) using DAB/peroxidase and GFAP (1:200, Epitomics, Burlingame, CA, USA, 2301-1) with Vector blue/alkaline phosphatase using the same method.

Techniques: Expressing, Infection, Immunostaining, Western Blot, Immunohistochemistry

Journal: Oncotarget

Article Title: Menin mediates Tat-induced neuronal apoptosis in brain frontal cortex of SIV-infected macaques and in Tat-treated cells

doi: 10.18632/oncotarget.14993

Figure Lengend Snippet: Menin expression (dark blue) is mainly observed in the nuclei of neurons (NeuN brown) in the frontal cortex of SIV-infected macaques A . Menin (dark blue) is also positive in activated microglial cytoplasm and processes, but not in microglial nuclei (Iba1, red) B . Menin (brown) is rarely stained in astrocytes of the cerebral cortex (GFAP, dark blue) C . but is positive in membranes and processes of white matter astrocytes (GFAP, dark blue) D . Representative double-labeled immunofluorescence images show NeuN (green) and menin (red) expression in the frontal cortex E–F . Menin expression is increased in SIV-infected macaques (E) compared with control macaques (F). Analysis of IOD of the double-positive area (yellow) shows significantly increased menin expression in neuronal nuclei of the SIV-infected macaques ( #2, 4, 7, 8 ) compared with control macaques ( #10–13 ) G . Data are expressed as mean ± SD, * P < 0.05. Original magnification: (A–D) 800×, (E–F) 400×. (A) From macaque #7 ; (B) from macaque #1; (C, D) from macaque #3 . (E) from macaque #7 ; (F) from macaque #13 . Arrows showing positive cells of double IHC staining.

Article Snippet: We also performed double-labeled IHC against menin (1:200, rabbit, Cell Signaling Technology, MA, USA, #6891) using DAB/peroxidase and GFAP (1:200, Epitomics, Burlingame, CA, USA, 2301-1) with Vector blue/alkaline phosphatase using the same method.

Techniques: Expressing, Infection, Staining, Labeling, Immunofluorescence, Immunohistochemistry

Journal: Oncotarget

Article Title: Menin mediates Tat-induced neuronal apoptosis in brain frontal cortex of SIV-infected macaques and in Tat-treated cells

doi: 10.18632/oncotarget.14993

Figure Lengend Snippet: Astrocytic gliosis is shown in the frontal cortex with GFAP IHC A . TUNEL-positive cells B . and ssDNA-positive cells C . are mainly small neuronal and glial cells. Arrows showing positive cells of IHC staining (A, B, C). Apoptosis of pyramidal neurons and small neurons of the cortex is seen by double-labeled IHC for ssDNA (blue) and NeuN (brown, D ). Menin- (brown) and cleaved-caspase 3- (dark blue) double-labeled cells E . Arrows showing positive cells of double IHC staining D, E . Significantly positive correlation ( P = 0.0118, R = 0.6722) is demonstrated between the number of cleaved-caspase 3-positive cells and menin-positive cells in 13 macaques F . There is a significant negative correlation between the number of NeuN-positive cells and menin-positive cells in 13 macaques ( P = 0.0069, R = −0.707) G . Original magnification: (A–E) 400×. (A) From macaque #9 ; (B-C) from macaque #6 ; (D) from #5 ; (E) from #4 .

Article Snippet: We also performed double-labeled IHC against menin (1:200, rabbit, Cell Signaling Technology, MA, USA, #6891) using DAB/peroxidase and GFAP (1:200, Epitomics, Burlingame, CA, USA, 2301-1) with Vector blue/alkaline phosphatase using the same method.

Techniques: TUNEL Assay, Immunohistochemistry, Labeling

Journal: Oncotarget

Article Title: Menin mediates Tat-induced neuronal apoptosis in brain frontal cortex of SIV-infected macaques and in Tat-treated cells

doi: 10.18632/oncotarget.14993

Figure Lengend Snippet: SH-SY5Y cells were transfected with pRK5M-Tat-flag or pRK5M-flag for 24 hours and subjected to IHC staining. Menin (green) and Tat (red) co-localized in the nuclei of SH-SY5Y cells A . Optical density assay showed that menin expression is significantly increased in SH-SY5Y cells transfected with pRK5M-Tat-flag compared with the group transfected with pRK5M-flag B . Representative western blot were shown C . Optical density analysis of menin expression D . Optical density analysis of cleaved caspase3 expression E . Primary neurons were treated with or without Tat (100 ng/mL) for 48 h. Menin and cleaved-caspase 3 is significantly increased in Tat-treated neurons compared with controls F-H . TUNEL staining shows significantly increased apoptosis (green) in pRK5M-Tat-flag-transfected SH-SY5Y cells I-J . Data are plotted as mean ± SD (n = 3). * P < 0.05, ** P < 0.01.

Article Snippet: We also performed double-labeled IHC against menin (1:200, rabbit, Cell Signaling Technology, MA, USA, #6891) using DAB/peroxidase and GFAP (1:200, Epitomics, Burlingame, CA, USA, 2301-1) with Vector blue/alkaline phosphatase using the same method.

Techniques: Transfection, Immunohistochemistry, Expressing, Western Blot, TUNEL Assay, Staining

Journal: Nanoscale

Article Title: Differentiating sepsis from non-infectious systemic inflammation based on microvesicle-bacteria aggregation.

doi: 10.1039/c5nr01851j

Figure Lengend Snippet: Figure 1: Trigger-dependent Microvesicle Shedding. Scanning electron micrograph (a) and size-distribution assessed by NTA (b) of PMN-derived microvesicles originating from PMNs incubated with plasma-opsonized S. aureus bacteria, E. coli, LPS, heat-inactivated bacteria bioparticles or vehicle (HBSS). PMN-derived CD11β/CD18 and CD11β/CD177-double positive events assessed by flow cytometry as a function of bacterial triggering agent (n = 3) (c). Scanning (d,e) and transmission electron micrographs (f,g) of PMNs showing pronounced membrane budding and shedding of microvesicles following incubation with opsonised S. aureus particles for 30 minutes (arrow indicates S. aureus particle) (e,g) compared to PMNs incubated with HBSS (d,f). 3D-tomographies and outer surface reconstructions of PMN incubated with S. aureus further confirmed the constriction of vesicles from the outer membrane seen in TEM (h). Raman spectroscopy maps of PMN incubated with (top, I) or without (bottom, II, control) bacteria showed lipid droplets and peri-membranous accumulation of glycogen granules in stimulated PMNs (I) compared to control (II) (i). PMNs exposed to S. aureus compared to resting PMNs (Figure 1d,e). Transmission electron micrographs of thin sections of PMNs containing phagocytised S. aureus bacteria confirmed increased membrane budding and formation of microvesicles (Figure 1f,g). Formation of glycogen granule clusters, translocation and peri- membranous massing of glycogen granule aggregates, and shipping of cytoplasmatic microvesicles containing glycogen granules were observed in PMNs exposed to bacteria, while glycogen granules remained well-dispersed in the cytoplasm of unstimulated PMNs (Figure 1f,g). 3D-tomography of PMNs further confirmed

Article Snippet: For flow cytometry, FITC mouse anti-rat CD18 (WT3, IgG1, AbD Serotec) and Alexa-647 anti-rat CD11β (OX-42, IgG2a, κ, BioLegend) were used for double staining at a concentration of 1 μg mL−1.

Techniques: Derivative Assay, Incubation, Clinical Proteomics, Bacteria, Flow Cytometry, Transmission Assay, Membrane, Raman Spectroscopy, Control, Translocation Assay, Tomography

Journal: Nanoscale

Article Title: Differentiating sepsis from non-infectious systemic inflammation based on microvesicle-bacteria aggregation.

doi: 10.1039/c5nr01851j

Figure Lengend Snippet: Figure 3. Microvesicles in Plasma Samples from an Experimental Sepsis Model. Caecal ligation and puncture (CLP) procedure in rats (a). Time-dependent concentration of neutrophil- derived CD11β/CD18-double positive microvesicles assessed by flow cytometry (b). Aggregation of S. aureus bacteria standard with microvesicle isolates from animal plasma at the 24 and 48 hour time point (c) and corresponding ROC curves (d). Characterization of Microvesicle-Bacteria Aggregates In order to better understand the nature of the microvesicle-bacteria aggregates, we used an in vitro analysis to further characterize their properties. The CD11β-positivity of the aggregating human PMN- derived vesicles was confirmed by immunostaining (Figure 4a) and transmission electron micrographs of microvesicle-bacteria aggregates were recorded (Figure 4b). The microvesicle- concentration dependence of bacteria aggregation was confirmed by serially diluting microvesicle isolates from PMNs exposed to S.

Article Snippet: For flow cytometry, FITC mouse anti-rat CD18 (WT3, IgG1, AbD Serotec) and Alexa-647 anti-rat CD11β (OX-42, IgG2a, κ, BioLegend) were used for double staining at a concentration of 1 μg mL−1.

Techniques: Clinical Proteomics, Ligation, Concentration Assay, Derivative Assay, Flow Cytometry, Bacteria, In Vitro, Immunostaining, Transmission Assay

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: Laboratory investigation; a journal of technical methods and pathology

Article Title: RIP1/RIP3/MLKL mediates dopaminergic neuron necroptosis in a mouse model of Parkinson disease.

doi: 10.1038/s41374-019-0319-5

Figure Lengend Snippet: Fig. 3 Double immunofluorescence staining showed colocalization and high expression of RIP1/RIP3/MLKL in SNpc at day 7 after MPTP treatment. a Immunostaining analysis of RIP1 in SNpc. b Immunostaining analysis of RIP3 in SNpc. c Immunostaining ana- lysis of MLKL in SNpc. a–c Scale bar = 20 μm. n = 6 mice per group

Article Snippet: Chicken polyclonal anti-TH primary antibody (1:1000, ab76442, Abcam, MA), rabbit polyclonal anti-RIP1 primary antibody (1:100, ab106393, Abcam, MA), rabbit polyclonal anti-RIP3 primary antibody (1:100, ab56164, Abcam, MA), rabbit polyclonal anti-MLKL primary antibody (1:100, 21066-1-AP, Proteintech, Wuhan, China), rabbit polyclonal anti-GFAP antibody (1:200, ab7260, Abcam, MA), rat anti-CD68 antibody (1:200, ab53444, Abcam, MA), Alexa 488-conjugated goat antichicken secondary antibody (1:200, ab150173, Abcam, MA) and Alexa 594-conjugated goat anti-rabbit secondary antibody (1:200, SA00006-3, Proteintech, Wuhan, China) were used to perform double immunofluorescence staining.

Techniques: Staining, Expressing, Immunostaining, Lysis

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: RIP1/RIP3/MLKL mediates dopaminergic neuron necroptosis in a mouse model of Parkinson disease.

doi: 10.1038/s41374-019-0319-5

Figure Lengend Snippet: Fig. 8 Schematic representation of potential mechanism of RIP1/RIP3/ MLKL-mediated DA neuron degeneration via regulation of necrop- tosis and neuroinflammation in SNpc after MPTP treatment in mice. DA neurons, dopaminergic neurons

Article Snippet: Chicken polyclonal anti-TH primary antibody (1:1000, ab76442, Abcam, MA), rabbit polyclonal anti-RIP1 primary antibody (1:100, ab106393, Abcam, MA), rabbit polyclonal anti-RIP3 primary antibody (1:100, ab56164, Abcam, MA), rabbit polyclonal anti-MLKL primary antibody (1:100, 21066-1-AP, Proteintech, Wuhan, China), rabbit polyclonal anti-GFAP antibody (1:200, ab7260, Abcam, MA), rat anti-CD68 antibody (1:200, ab53444, Abcam, MA), Alexa 488-conjugated goat antichicken secondary antibody (1:200, ab150173, Abcam, MA) and Alexa 594-conjugated goat anti-rabbit secondary antibody (1:200, SA00006-3, Proteintech, Wuhan, China) were used to perform double immunofluorescence staining.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: Aromatase (Aro) expression in astrocytes in glia-enriched subcultures. Cells were isolated from P0–P5 newborn rats. Aro (red) is strongly expressed in GFAP-positive astrocytes (green) and colocalizes with the nuclear staining (DAPI, blue). Fibrous astrocytes with small somata and long, thin unbranched processes ( a – h ) show strong nuclear Aro expression ( a , c , d , white arrowheads). Cytoplasmic Aro expression is weaker, and the long thin processes do not show Aro immunopositivity. Protoplasmic astrocytes ( i – p ) with bigger somata and short, thick and frequently branched processes also display nuclear Aro expression ( i , k , l , white arrowheads). Weak immunopositivity is detectable in the processes. The nuclear Aro signal distributes evenly or in a dotted pattern ( c , k , white arrowheads) or strongly concentrates to certain parts of the nucleus ( g ) in both astrocyte subtypes. (White arrowheads indicate a selected representative cell and its nucleus with the Aro signal). Scale bar: 100 μm.

Article Snippet: In spite of the limited availability of commercially available aromatase antibodies for studying its expression in the rat brain by immunohistochemistry, as highlighted by Krentzel et al. [ ], to date, the aromatase antibody from Novus Biologicals (B100-1596) was not applied for investigating Aro expression in the CNS.

Techniques: Expressing, Isolation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: Quantitative analysis of aromatase localization in astrocytes. Analysis of mean fluorescent intensity revealed that Aro intensity is significantly stronger in the nucleus than in the cytoplasm in different astrocyte subtypes. GFAP-positive fibrous astrocytes showed stronger nuclear Aro intensity (36.36 ± 7.7; n = 25) than GFAP-positive protoplasmic astrocytes (32.25 ± 9.18; n = 29). Nuclear Aro intensity in the S100b-positive subtype (21.72 ± 4.98; n = 24) was significantly lower than in the GFAP-positive subtype. Data are presented in mean ± SD. * p < 0.05, *** p < 0.001, ### p < 0.001. Asterisks indicate the significance between the nucleus and cytoplasm within each group. Pound (###) indicates the significantly lower intensity of nuclear Aro between GFAP and S100b positive astrocyte subtypes.

Article Snippet: In spite of the limited availability of commercially available aromatase antibodies for studying its expression in the rat brain by immunohistochemistry, as highlighted by Krentzel et al. [ ], to date, the aromatase antibody from Novus Biologicals (B100-1596) was not applied for investigating Aro expression in the CNS.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: Localization of aromatase in astrocytes in adult rat brain sections. Nuclear (nucleus: blue) appearance of Aro enzyme (red; c , g , k , o ; yellow arrowheads) in GFAP-labelled (green) astrocytes ( a , b , e , f , i , j , m , n ; white arrowheads) is also detectable in frozen cortical tissue sections from male ( a – h ) and female ( i – p ) rats. Interestingly, the Aro immunopositive signal in the adult cortical astrocytes is abundant in the processes ( c , g , k , o ; green arrowheads), while astrocytes derived from newborn rats do not show an Aro signal in the branches. Quantitative analysis of total GFAP-positive astrocytes and nuclear Aro immunopositive astrocytes ( q ) in 10 randomly selected microscopic fields of different frozen sections from both sexes showed that the abundance of nuclear Aro immunopositive GFAP-labelled astrocytes is about 20% in both males (19.79%; n = 197) and females (20.13%; n = 154). Scale bar: 100 μm.

Article Snippet: In spite of the limited availability of commercially available aromatase antibodies for studying its expression in the rat brain by immunohistochemistry, as highlighted by Krentzel et al. [ ], to date, the aromatase antibody from Novus Biologicals (B100-1596) was not applied for investigating Aro expression in the CNS.

Techniques: Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: Localization of aromatase in microglia cells. CD11b/c (Ox42)-labelled microglia cells (green) isolated from newborn rats show Aro (red) expression, but the signal is detectable exclusively in the cytoplasm. Microglia cells with typical ameboid/activated morphology ( a – h ) show stronger Aro expression compared with the resting/ramified forms ( i – p ), where Aro immunopositivity is much weaker. Aro signal is weak or undetectable in the branches of ramified microglia cells. Quantitative analysis of the fluorescent intensity confirmed that the Aro signal is significantly higher in the cytoplasm of ameboid cells (18.64 ± 2.81; n = 15) than in ramified cells (9.98 ± 1.35; n = 15) ( r ). Although Aro is not presented in the nucleus of microglia cells, a low level of fluorescent intensity was detectable in the nuclear area due to the overprojection of staining of neighbouring cells from the upper and lower cellular layers and/or the overlapping of cytoplasmic signal, but its level was significantly lower (9.71 ± 2.31; n = 45) than the cytoplasmic Aro intensity (14.67 ± 4.68; n = 45). The analysis of mean fluorescent intensity also revealed that the level of Aro immunopositivity in the cytoplasm is similar in astrocytes (14.20 ± 4.9; n = 54) and microglia cells ( q ). Data are presented in mean ± SD. ** p < 0.01, *** p < 0.001, ### p < 0.001. Asterisks indicate the significance between microglia cells. Pound (###) indicates the significance between astrocytes and microglia. Scale bar: 100 μm.

Article Snippet: In spite of the limited availability of commercially available aromatase antibodies for studying its expression in the rat brain by immunohistochemistry, as highlighted by Krentzel et al. [ ], to date, the aromatase antibody from Novus Biologicals (B100-1596) was not applied for investigating Aro expression in the CNS.

Techniques: Isolation, Expressing, Staining

Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: Nuclear aromatase expression overlaps with nuclear ERα signal. Double immunostaining with ERα (green) and Aro (red) in glia culture ( a – l ) shows that nuclear Aro signals colocalize with nuclear ERα ( a , e , i ; white arrowheads). Both Aro and ERα are strongly represented in the nucleus ( b , c , f , g , j,k ; yellow arrowheads) and also in the cytoplasm ( b , c , f , g , j , k ; blue arrowheads). ERα positive dots are also detectable in the processes ( b , f,j ; orange arrowheads). Scale bar: 100 μm.

Article Snippet: In spite of the limited availability of commercially available aromatase antibodies for studying its expression in the rat brain by immunohistochemistry, as highlighted by Krentzel et al. [ ], to date, the aromatase antibody from Novus Biologicals (B100-1596) was not applied for investigating Aro expression in the CNS.

Techniques: Expressing, Double Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: Localization of aromatase in the rat ovary and in human granulosa cells. Immunostaining of rat ovarian tissue sections is presented in images ( a – f ). Immunohistochemistry revealed that Aro (red) is strongly expressed in the whole ovarian tissue during the oestrus cycle but does not colocalize with the nuclear signal (blue). Results from Aro-stained human granulosa cells (red) corroborate the immunohistochemical findings as Aro immunopositivity is abundant in granulosa cells but only in the cytoplasm ( g – l ). Scale bar: 100 μm.

Article Snippet: In spite of the limited availability of commercially available aromatase antibodies for studying its expression in the rat brain by immunohistochemistry, as highlighted by Krentzel et al. [ ], to date, the aromatase antibody from Novus Biologicals (B100-1596) was not applied for investigating Aro expression in the CNS.

Techniques: Immunostaining, Immunohistochemistry, Staining, Immunohistochemical staining

Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: The simplified structure of human aromatase (Aro) protein with important conserved regions shown in red (based on Di Nardo et al. ). Blue lines indicate which parts of the human Aro protein were used to produce antibodies for Aro detection in CNS studies . Green line indicates a mouse Aro peptide to generate an anti-mouse Aro antibody. Co: commercially available antibodies; In-h: antibodies made and validated in-house; * the Novus Aro antibody used in this study.

Article Snippet: In spite of the limited availability of commercially available aromatase antibodies for studying its expression in the rat brain by immunohistochemistry, as highlighted by Krentzel et al. [ ], to date, the aromatase antibody from Novus Biologicals (B100-1596) was not applied for investigating Aro expression in the CNS.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: List of primary antibodies used in this study. According to the technical information provided by the manufacturers, the commercial primary antibodies used in the study were verified by Knockdown or Relative expression to ensure that the antibody binds to the antigen stated.

Article Snippet: In spite of the limited availability of commercially available aromatase antibodies for studying its expression in the rat brain by immunohistochemistry, as highlighted by Krentzel et al. [ ], to date, the aromatase antibody from Novus Biologicals (B100-1596) was not applied for investigating Aro expression in the CNS.

Techniques: Knockdown, Expressing, Binding Assay, Recombinant