Journal: Gene therapy

Article Title: Targeted inhibition of platelet-derived growth factor receptor-beta subunit in hepatic stellate cells ameliorates hepatic fibrosis in rats.

doi: 10.1038/gt.2008.93

Figure Lengend Snippet: Figure 3 Localization of LacZ driven by CMV promoter and GFAP promoter in normal rats, acute liver injury rats and chronic liver injury rats. (a) Representative graphs of b-galactosidase (b-gal) immunofluorescence showed the distribution of b-gal-positive cells in normal rats, acute liver injury rats and chronic liver injury rats treated with pCMV-shRNA-LacZ or pGfa-shRNA-LacZ. Magnification of 20. The scale bar represents 80 mm. (b) Representative graphs of a-SMA and GFAP immunofluorescence showed that in the chronic injured liver, a-SMA and GFAP proteins distributed mainly around the portal area and the hyperplastic bile duct, whereas in the acute injured liver, both the proteins revealed a much more diffuse distribution in the hepatic lobule. In addition, the GFAP-positive cells were more than the a-SMA- positive cells both in the acute injured liver and in the chronic injured liver. Magnification of 20. The scale bar represents 80 mm. (c) Representative graphs of b-gal and a-SMA double-staining revealed that b-gal protein in pCMV-shRNA-LacZ-treated livers could be expressed in hepatocytes (blue arrow) and activated HSCs stained by a-SMA (white arrow), magnification of 63. The scale bar represents 40 mm. (d) Representative graphs of b-gal and GFAP double-staining showed that b-gal-positive cells were all GFAP-positive HSCs (pink arrow) in pGfa-shRNA-LacZ-treated livers, magnification of 63. The scale bar represents 40 mm. (e) Representative graphs of b-gal and a-SMA double-staining showed that some b-gal protein was expressed in activated HSCs stained by a-SMA (white arrow) in pGfa-shRNA- LacZ-treated livers, magnification of 63. The scale bar represents 40 mm. CMV, cytomegalovirus; GFAP, glial fibrillary acidic protein; HSCs, hepatic stellate cells; PDGFR-b, platelet-derived growth factor receptor-b subunit; a-SMA, a-smooth muscle actin; shRNA, short hairpin RNA; RT-PCR, reverse transcriptional-PCR.

Article Snippet: Frozen liver sections (8 mm in thickness) were incubated with mouse anti-b-galactosidase monoclonal antibody (Promega, Madison, WI, USA), rabbit anti-GFAP polyclonal antibody (Boster, Wuhan, China) or rabbit anti-a-SMA polyclonal antibody (Bios, Beijing, China) for 20 h at 4 1C, then washed in phosphate-buffered saline and incubated for 30 min with Cy5-labeled anti-rabbit IgG antiserum and FITC-labeled anti-mouse IgG antiserum (Jackson, West Grove, PA, USA) at 1:100 dilution in phosphate-buffered saline.

Techniques: shRNA, Double Staining, Staining, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of tissue science & engineering

Article Title: Whole-Cell Dissociated Suspension Analysis in Human Brain Neurodegenerative Disease: A Pilot Study

doi:

Figure Lengend Snippet: Inorganic and organic supplements.

Article Snippet: , Mouse anti-GFAP (Clone 1B4) Alexa Fluor 647 , Cow spinal cord homogenate , BD Pharmingen/560298.

Techniques: Marker, Recombinant, Purification, Residue, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Bisphenol AF Induced Neurodevelopmental Toxicity of Human Neural Progenitor Cells via Nrf2/HO-1 Pathway

doi: 10.3390/ijms26125685

Figure Lengend Snippet: BPAF reduced the number of neurons and injured synaptic growth. ( A ) Representative immunofluorescence images of ReNcell CX cells stained with the newborn neuronal marker doublecortin (DCX, Green), while the nuclear stain was DAPI (Hoechst 33342, blue) (Scale bar, 200 μm). ( C ) Sholl analysis of the number of intersections of radial distance from the cell body (N = 30 cells). ( D ) Representative immunofluorescence images of ReNcell CX cells stained with the astrocytes marker (GFAP), and the nuclei were counterstained by DAPI (blue) (Scale bar, 200 μm). ( B , E ) The percentage of immunofluorescence-positive cells. One-way ANOVA was applied to test the difference between two or more groups; two-way ANOVA and Bonferroni post-test were applied for the results with Sholl analysis. The data presented here represent the mean ± SD of at least three separate experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 when compared to the corresponding control group.

Article Snippet: The primary and secondary antibodies are anti-rabbit DCX, anti-mouse GFAP (1:200, Cell Signaling Technology, Danvers, MA, USA), and Alexa Fluor GFAP-labeled goat anti-rabbit antibody (1:200, Beyotime, Haimen, China), respectively.

Techniques: Immunofluorescence, Staining, Marker, 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: Nature Communications

Article Title: Optochemical control of slow-wave sleep in the nucleus accumbens of male mice by a photoactivatable allosteric modulator of adenosine A 2A receptors

doi: 10.1038/s41467-024-47964-4

Figure Lengend Snippet: a Schematic of pharmacologic activation of NAc by focal injection of adenosine, CGS 21680, CPA, or ZM 241385 into freely behaving WT and A 2A R KO mice, illustrated by Sara Kobayashi. b Total amount of SWS for 5 h after focal drug injection into the NAc. Data [left to right, n = 6 (Veh), 4 (AD), 6 (CGS), 6 (CPA), 4 (Veh), 4 (ZM), 5 (Veh), and 5 (AD) biologically independent animals in each group] are presented as mean ± SEM. Unpaired 2-tailed t-test compared with the vehicle injections. c Fluorescence image showing a representative example of the injection site with fluorescein located in the NAc. The experiment was independently repeated 2 times. d Schematic of AAV microinjection and placement of microdialysis probe in the NAc of WT or A 2A R KO mice, illustrated by Sara Kobayashi. e Immunostaining for NeuN (upper panel) or GFAP (lower panel) together with mCherry in WT mice injected with an AAV expressing a hM3Dq DREADD/mCherry fusion protein under the GFAP promoter (AAV-GFAP-hM3DqDREADD). The experiment was independently repeated 6 (WT) and 5 (A 2A R KO) times. Scale bar: 20 μm. f Drawings of superimposed AAV-GFAP-hM3DqDREADD injection sites in the NAc core of WT (in red) and A 2A R KO (in blue) mice are shown. Time course ( g , h ) and total amount ( i ) of SWS after chemogenetic stimulation of astrocytes in the NAc of WT ( g, i ) and A 2A R KO ( h, i ) mice. Data [n = 6 ( g , WT groups in i ) and n = 5 ( h , A 2A R KO groups in i ) biologically independent animals in each group] are presented as mean ± SEM. Unpaired 2-tailed t-test compared with vehicle (saline) injection. j Typical implantation site for the guide cannula and location of the microdialysis probe in the NAc. Immunostaining for mCherry indicates the AAV-infected area in the NAc. Scale bar: 400 μm. k Extracellular adenosine levels normalized to vehicle (saline) injections in the NAc of WT mice injected with AAV-GFAP-mCherry, AAV-GFAP-hM3DqDREADD or AAV-hSyn-hM3DqDREADD, A 2A R KO mice injected with AAV-GFAP-hM3DqDREADD. Data (n = 4 biologically independent animals/group) are presented as mean ± SEM. Unpaired 2-tailed t-test compared with AAV-GFAP-mCherry-injected mice. Source data have been deposited in the Figshare database [10.6084/m9.figshare.25468084]. Abbreviations: AAV adeno-associated virus, AD adenosine, A 2A R adenosine A 2A receptor, CNO clozapine N-oxide, DREADD designer receptors exclusively activated by designer drugs, EEG electroencephalogram, GFAP glial fibrillary acidic protein, hSyn human synapsin, KO knockout, LV lateral ventricle, NAc nucleus accumbens, NeuN neuronal nuclei, ns not significant, pA polyadenylation signal, SEM standard error of the mean, SWS slow-wave sleep, Veh vehicle, WPRE woodchuck hepatitis virus posttranscriptional regulatory element, WT wild type.

Article Snippet: The sections were then incubated with the rabbit anti-GFAP antibody (1:200, Cat#HPA056030, Merck), goat anti-mCherry antibody (1:1000, Cat# AB0040-200, SICGEN), mouse anti-NeuN antibody (1:100, Cat# MAB377, Millipore) containing 5% BlockAce at room temperature in the combinations described in the Results section.

Techniques: Activation Assay, Injection, Fluorescence, Immunostaining, Expressing, Saline, Infection, Virus, Knock-Out