Journal: Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society

Article Title: Altered gastrointestinal motility involving autoantibodies in the experimental autoimmune encephalomyelitis model of multiple sclerosis

doi: 10.1111/nmo.13349

Figure Lengend Snippet: The area of cells in the ENS was calculated in SJL mice immunized with MSCH EAE or control mice. Three different cell markers were used. (A), There was no difference in the percentage of pixels per ganglion that expressed neuron HuD, (control n = 5, EAE n = 5, n.s.) B) There was no difference in the percentage of pixels per ganglion that expressed the glial protein S100β, (control n = 5, EAE n = 5, n.s.) C) There was a significant reduction in the distribution of GFAP within ENS ganglia of EAE mice compared to control mice (control n = 5, EAE n = 5, P < .012). Data represented as mean ± SEM and analyzed by 2-tailed Welch’s t test. Five ganglia were imaged per animal. Representative images demonstrate the distribution of HuD, S100β and GFAP in control mice (D, E, F) and EAE mice (G, H, I), respectively

Article Snippet: 2.6 | Mouse immunohistochemistry Longitudinal muscle and myenteric plexus (LMMP) preparations were dissected and stained for either HuD, GFAP, or S100β (1:100 rabbit anti-HuD IgG, Santa Cruz Biotechnology, 1:500 rabbit anti-GFAP, EnCor Biotechnology, or 1:1000 rabbit anti-S100β, Abcam) with a Cy3 conjugated goat anti-rabbit IgG secondary antibody (1:150, Jackson ImmunoResearch).

Techniques: Control

Journal: Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society

Article Title: Altered gastrointestinal motility involving autoantibodies in the experimental autoimmune encephalomyelitis model of multiple sclerosis

doi: 10.1111/nmo.13349

Figure Lengend Snippet: Patterns of serum immunoreactivity of MS and EAE serum against ENS structures of guinea pig LMMP. MS patients who experience constipation include glial-predominant and neuronal-predominant staining. (A) Example of an MS patient serum sample that co-localizes with S100β antibodies. (B) Example of an MS patient serum sample that co-localizes with HuD antibodies. Immunoreactivity of mouse serum against guinea pig LMMP. (C) Naïve B6 control mice (top) and CFA-treated B6 control mice (bottom) do not exhibit positive immunoreactivity against cells of the ENS. D) EAE B6 + MOG-treated mice demonstrated immunoreactivity against ENS neurons (top) or against neurons and glia (bottom). Images shown at 20x magnification

Article Snippet: 2.6 | Mouse immunohistochemistry Longitudinal muscle and myenteric plexus (LMMP) preparations were dissected and stained for either HuD, GFAP, or S100β (1:100 rabbit anti-HuD IgG, Santa Cruz Biotechnology, 1:500 rabbit anti-GFAP, EnCor Biotechnology, or 1:1000 rabbit anti-S100β, Abcam) with a Cy3 conjugated goat anti-rabbit IgG secondary antibody (1:150, Jackson ImmunoResearch).

Techniques: Staining, Control

Journal: Biomarker Insights

Article Title: Characterization of Antibodies that Detect Human GFAP after Traumatic Brain Injury

doi: 10.4137/BMI.S9873

Figure Lengend Snippet: Lysates from the indicated sources were digested with increasing amounts of rat capn2 (triangles) and probed with the antibodies labeled on the right. Spectrin served as a positive control for digestion efficiency, and was cleaved to SBDP150 and SBDP145 in a capn2 concentration-dependent manner. ( A ) In lysates from human HEK293 cells over-expressing human GFAP, both antibodies detected GFAP from 50–38 kDa, with capn2 cleaving GFAP to a 38 kDa limit fragment. ( B ) The anti-GFAP Detection antibody recognized full length endogenous GFAP and capn2-generated GFAP-BDPs from rat and mouse brain, while the Capture antibody did not.

Article Snippet: The anti-GFAP rabbit IgG polyclonal antibody (Detection antibody) was made by immunizing rabbits with purified full length recombinant human GFAP protein (Banyan) at Pocono Rabbit Farm and Laboratory (Pocono) as previously described by Papa et al.

Techniques: Labeling, Positive Control, Concentration Assay, Expressing, Generated

Journal: Biomarker Insights

Article Title: Characterization of Antibodies that Detect Human GFAP after Traumatic Brain Injury

doi: 10.4137/BMI.S9873

Figure Lengend Snippet: ( A ) Control (normal, non head-injured) human post-mortem brain lysate (20 μg/lane) was separated by SDS-PAGE, blotted, and probed in parallel with the anti-GFAP Capture and Detection antibodies, which yielded similar banding patterns from 50–38 kDa. ( B ) Recombinant full length human GFAP (rhGFAP) was purified from E. coli , and then 4 μg was loaded and visualized with Coomassie stain. Also, 50 ng/lane was loaded, and blots were probed with the anti-GFAP Capture or Detection antibodies, which showed predominantly 50 kDa protein. ( C ) rhGFAP was cut or not with rat calpain-2 (capn2), then blotted at 50 ng/lane and visualized with the anti-GFAP Detection antibody. Capn2 digestion of rhGFAP yielded bands from 50–38 kDa.

Article Snippet: The anti-GFAP rabbit IgG polyclonal antibody (Detection antibody) was made by immunizing rabbits with purified full length recombinant human GFAP protein (Banyan) at Pocono Rabbit Farm and Laboratory (Pocono) as previously described by Papa et al.

Techniques: Control, SDS Page, Recombinant, Purification, Staining

Journal: Biomarker Insights

Article Title: Characterization of Antibodies that Detect Human GFAP after Traumatic Brain Injury

doi: 10.4137/BMI.S9873

Figure Lengend Snippet: Protein lysates from the indicated human organs (20 μg/lane) were blotted and probed with the indicated antibodies. Both the Capture ( A ) and Detection ( B ) antibodies recognized GFAP from 50–38 kDa exclusively in the brain.

Article Snippet: The anti-GFAP rabbit IgG polyclonal antibody (Detection antibody) was made by immunizing rabbits with purified full length recombinant human GFAP protein (Banyan) at Pocono Rabbit Farm and Laboratory (Pocono) as previously described by Papa et al.

Techniques:

Journal: Biomarker Insights

Article Title: Characterization of Antibodies that Detect Human GFAP after Traumatic Brain Injury

doi: 10.4137/BMI.S9873

Figure Lengend Snippet: Equal aliquots of CSF drawn at one day after injury from 8 severe human TBI patients were blotted and probed with the anti-GFAP Detection antibody. CSF from one representative control patient is also shown. Full length GFAP and GFAP-BDP were detected from 50–38 kDa. All samples were run together on the same gel/blot; intervening lanes were removed.

Article Snippet: The anti-GFAP rabbit IgG polyclonal antibody (Detection antibody) was made by immunizing rabbits with purified full length recombinant human GFAP protein (Banyan) at Pocono Rabbit Farm and Laboratory (Pocono) as previously described by Papa et al.

Techniques: Control, Western Blot

Journal: Biomarker Insights

Article Title: Characterization of Antibodies that Detect Human GFAP after Traumatic Brain Injury

doi: 10.4137/BMI.S9873

Figure Lengend Snippet: Immunoprecipitations (IP) were performed with biotinylated anti-GFAP Capture antibody and streptavidin (SA)-coupled beads (Capture SA IP) or with SA beads only as a negative control (SA IP only). GFAP was immunoprecipitated separately from two sources, human post-mortem brain lysate ( A ) or CSF pooled from 3 severe TBI patients ( B ). Samples of brain lysate or CSF were resolved prior to IP (pre lane 1), and after IP (post lanes 2, 5). Proteins were eluted from the SA beads (eluate lanes 3, 6), and then the beads were washed with SDS-PAGE buffer (wash lane 4). Blots were probed with anti-GFAP Detection antibody. ( A and B ). Biotinylated anti-GFAP Capture antibody recovered full length GFAP and GFAP-BDP from human brain lysate and TBI CSF, without appearing to enrich for any particular GFAP band.

Article Snippet: The anti-GFAP rabbit IgG polyclonal antibody (Detection antibody) was made by immunizing rabbits with purified full length recombinant human GFAP protein (Banyan) at Pocono Rabbit Farm and Laboratory (Pocono) as previously described by Papa et al.

Techniques: Negative Control, Immunoprecipitation, SDS Page

Journal: Molecular Therapy. Nucleic Acids

Article Title: Silencing of the MEKK2/MEKK3 Pathway Protects against Spinal Cord Injury via the Hedgehog Pathway and the JNK Pathway

doi: 10.1016/j.omtn.2019.05.014

Figure Lengend Snippet: BBB Score and H&E Staining Confirmed the Successful Establishment of an SCI Rat Model (A) Motor function of rat hindlimbs at days 0, 7, 14, 21, and 28 after surgery evaluated according to the BBB scale. (B) Histopathological changes in spinal cords of rats at days 0, 7, 14, 21, and 28 after surgery detected by H&E staining (×400). (C and D) The positive levels of GFAP (C) and NF-200 (D) and in spinal cord tissues of rats at day 28 after surgery, evaluated by immunohistochemistry (×400). *p < 0.05 versus the normal group. The data are expressed as the mean ± SD. Data in (A) were compared by repeated-measures ANOVA and in (B)–(D) by unpaired t test; n = 10.

Article Snippet: After blocking, three randomly selected sections in each group were incubated with the mouse monoclonal anti-NF-200 antibody (1:100) and rabbit polyclonal anti-GFAP antibody (AmyJet Scientific, Wuhan, China) at 4°C overnight.

Techniques: Staining, Immunohistochemistry

Journal: Molecular Therapy. Nucleic Acids

Article Title: Silencing of the MEKK2/MEKK3 Pathway Protects against Spinal Cord Injury via the Hedgehog Pathway and the JNK Pathway

doi: 10.1016/j.omtn.2019.05.014

Figure Lengend Snippet: Suppression of the Hh Pathway Prevents SCI from Recovery (A) mRNA expression of Gli-1, Nestin, Smo, and Shh in the sham injury and SCI groups at day 28 after surgery, determined by qRT-PCR. (B) Protein levels of Gli-1, Nestin, Smo, and Shh in the sham injury and SCI groups at day 28 after surgery, determined by western blot analysis. (C) mRNA expression of Gli-1, Nestin, Smo, and Shh in each group at day 28 after surgery, determined by qRT-PCR. (D) Protein levels of Gli-1, Nestin, Smo, and Shh in each group at day 28 after surgery, determined by western blot analysis. (E) Motor function of rats’ hindlimbs in each group at days 0, 7, 14, 21, and 28 after transduction, evaluated by BBB scale. (F) Histopathological changes of spinal cord tissues in each group at day 0, 7, 14, 21, and 28 after transduction was detected by H&E staining (×100). (G) Positive levels of NF-200 and GFAP in each group at day 28 after transduction, determined by immunohistochemistry (×400). *p < 0.05 versus the sham injury/SCI+DMSO group. The data are expressed as the mean ± SD. Data in (E) were compared by repeated-measures ANOVA and in the other panels by unpaired t test; n = 10.

Article Snippet: After blocking, three randomly selected sections in each group were incubated with the mouse monoclonal anti-NF-200 antibody (1:100) and rabbit polyclonal anti-GFAP antibody (AmyJet Scientific, Wuhan, China) at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transduction, Staining, Immunohistochemistry

Journal: Molecular Therapy. Nucleic Acids

Article Title: Silencing of the MEKK2/MEKK3 Pathway Protects against Spinal Cord Injury via the Hedgehog Pathway and the JNK Pathway

doi: 10.1016/j.omtn.2019.05.014

Figure Lengend Snippet: Disruption of the JNK Pathway Has Protective Effects against SCI (A) mRNA expressions of MCP-1, MIP-1β, and MIP-3α in the sham injury and SCI groups at day 28 after surgery, determined by qRT-PCR. (B) Protein levels of p-JNK/JNK and p-c-Jun/c-Jun in the sham injury and SCI groups at day 28 after surgery, detected by western blot analysis. (C) mRNA expression of MCP-1, MIP-1β, and MIP-3α in each group at day 28 after surgery detected by qRT-PCR. (D) Protein levels of p-JNK/JNK and p-c-Jun/c-Jun in each group at day 28 after surgery, detected by western blot analysis. (E) Motor function of rat hindlimbs in each group at days 0, 7, 14, 21, and 28 after transduction evaluated by BBB rating. (F) Histopathological changes of spinal cord tissues in each group at day 28 after transduction determined by H&E staining (×100). (G) The positive levels of NF-200 and GFAP proteins in each group at day 28 after transduction, determined by immunohistochemistry (×400). *p < 0.05 versus the sham/SCI+DMSO group. The data are expressed as the mean ± SD. Data in (E) were compared by repeated-measures ANOVA and in the other panels by unpaired t test; n = 10.

Article Snippet: After blocking, three randomly selected sections in each group were incubated with the mouse monoclonal anti-NF-200 antibody (1:100) and rabbit polyclonal anti-GFAP antibody (AmyJet Scientific, Wuhan, China) at 4°C overnight.

Techniques: Disruption, Quantitative RT-PCR, Western Blot, Expressing, Transduction, Staining, Immunohistochemistry

Journal: Molecular Therapy. Nucleic Acids

Article Title: Silencing of the MEKK2/MEKK3 Pathway Protects against Spinal Cord Injury via the Hedgehog Pathway and the JNK Pathway

doi: 10.1016/j.omtn.2019.05.014

Figure Lengend Snippet: Overexpressed MEKK2/MEKK3 Aggravates SCI by Inhibiting the Hh Pathway and Activating the JNK Pathway (A) The transduction efficiency of MEKK2/MEKK3 vectors, determined by western blot analysis. (B) mRNA expression of Hh- and JNK-pathway-related factors in each group at day 28 after surgery, detected using qRT-PCR. (C) Protein levels of Hh-pathway-related factors in each group at day 28 after surgery, measured by western blot analysis. (D) Protein levels of JNK pathway-related factors in each group at day 28 after surgery, detected using western blot analysis. (E) Motor function of the hindlimbs of rats in each group at days 0, 7, 14, 21, and 28 after transduction, tested according to the BBB scale. (F) Histopathologic changes in spinal cord tissues in each group at day 28 after transduction, observed with H&E staining (×100). (G) Positive level of NF-200 and GFAP proteins in each group at day 28 after transduction, determined by immunohistochemistry (×400). *p < 0.05 versus the SCI+NC-vector group. The data are expressed as the mean ± SD. Data in (E) were compared by repeated-measures ANOVA and in the other panels by one-way ANOVA; n = 10.

Article Snippet: After blocking, three randomly selected sections in each group were incubated with the mouse monoclonal anti-NF-200 antibody (1:100) and rabbit polyclonal anti-GFAP antibody (AmyJet Scientific, Wuhan, China) at 4°C overnight.

Techniques: Transduction, Western Blot, Expressing, Quantitative RT-PCR, Staining, Immunohistochemistry, Plasmid Preparation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Silencing of the MEKK2/MEKK3 Pathway Protects against Spinal Cord Injury via the Hedgehog Pathway and the JNK Pathway

doi: 10.1016/j.omtn.2019.05.014

Figure Lengend Snippet: MEKK2/MEKK3 Disruption Alleviates SCI by Activating the Hh Pathway and Inhibiting the JNK Pathway (A) The transduction efficiency of si-MEKK2/MEKK3, determined by western blot analysis. (B) mRNA expression of Hh- and JNK pathway-related factors in each group at day 28 after transduction, evaluated by qRT-PCR. (C) Protein levels of Hh pathway-related factors in each group at day 28 after transduction, measured by western blot analysis. (D) Protein levels of JNK pathway-related factors in each group at day 28 after transduction, measured by western blot analysis. (E) motor function of the hindlimbs of rats in each group at days 0, 7, 14, 21, and 28 after transduction, evaluated according to the BBB scale. (F) Histopathological changes in spinal cord of rats in each group at day 28 after transduction, according to H&E staining (×400). (G) Positive level of NF-200 and GFAP proteins in each group at day 28 after transduction, determined by immunohistochemistry (×400). *p < 0.05 versus the SCI+si-NC group; # p < 0.05 versus the SCI+si-MEKK2/MEKK3+DMSO group. The data are expressed as the mean ± SD. Data in (E) were compared by repeated-measures ANOVA and in the other panels by one-way ANOVA; n = 10.

Article Snippet: After blocking, three randomly selected sections in each group were incubated with the mouse monoclonal anti-NF-200 antibody (1:100) and rabbit polyclonal anti-GFAP antibody (AmyJet Scientific, Wuhan, China) at 4°C overnight.

Techniques: Disruption, Transduction, Western Blot, Expressing, Quantitative RT-PCR, Staining, Immunohistochemistry