PA1-10004

antibody from Invitrogen Antibodies

Targeting: GFAP FLJ45472

Provider product page for PA1-10004

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [26]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [1]
Immunohistochemistry [1]
Other assay [19]

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Product number: PA1-10004 - Provider product page
Provider: Invitrogen Antibodies
Product name: GFAP Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Purifed from natural sources
Description: PA1-10004 was made against full length human recombinant GFAP, and works well on western blots, and on immunostaining of cell culture or tissue sections.
Concentration: Conc. not determined

GFAP protein structure - PA1-10004 shown in red.

Supportive validation

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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Optogenetic stimulation enhances neuronal and oligodendrocytic but not astrocytic differentiation of NPCs. ChR2-NPCs were differentiated for seven days. ( A - C ) Representative images of immunofluorescence staining for beta-III-tubulin (green; A), oligodendrocyte transcription factor 2 (Olig2) (red; nuclear signal indicated with yellow arrows; B), and glial fibrillary acidic protein (GFAP) (red; C) for unstimulated (ChR2) and BL-stimulated ChR2-NPCs (ChR2 + BL). ( D ) Quantification of the percentage of neurons (beta-III-tubulin-positive cells). Data are expressed as mean +- SEM from three independent experiments (ChR2: 4.5 +- 0.4; ChR2 + BL: 10.9 +- 1) *** p < 0.001 vs. ChR2 determined by Mann-Whitney test. ( E ) Quantification of the percentage of oligodendrocytes (Olig2-positive cells). Data are expressed as mean +- SEM from three independent experiments (ChR2: 5.3 +- 0.9; ChR2 + BL: 9.5 +- 0.9) * p < 0.05, unpaired t -test. ( F ) Quantification of the percentage of astrocytes (GFAP-positive cells). Data are expressed as mean +- SEM from three independent experiments (ChR2: 56.47 +- 2.2; ChR2 + BL: 58.65 +- 2.7). Scale bars, 20 uM.

Supportive validation

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Experimental details: Figure 4 Optogenetic stimulation triggers morphological and functional shift in ChR2-NPC-derived astrocytes. Astrocyte morphology was determined after GFAP staining. ( A ) Representative images of immunofluorescence staining for GFAP (orange) and nuclear detection by 4',6-diamidino-2-phenylindole (DAPI) (blue) in unstimulated ChR2-NPCs (ChR2). Yellow square shows higher magnification for polygonal astrocytes. Scale bars, 20 uM. ( B ) Representative images of immunofluorescence staining for GFAP (orange) and nuclear detection by DAPI (blue) in BL-stimulated ChR2-NPCs (ChR2 + BL). Yellow square shows higher magnification for stellate astrocyte. Scale bars, 20 uM. ( C ) Quantification of the ratio of polygonal astrocytes in unstimulated (ChR2) and BL-stimulated ChR2-NPCs (ChR2 + BL) cultures. Data are expressed as mean +- SEM from three independent experiments (ChR2: 64.9 +- 2.3; ChR2 + BL: 48.4 +- 3.4). * p < 0.05 vs. ChR2 determined by unpaired t -test. ( D ) Quantification of the ratio of stellate astrocytes in unstimulated (ChR2) and BL-stimulated ChR2-NPCs (ChR2 + BL) cultures. Data are expressed as mean +- SEM from three independent experiments (ChR2: 35.1 +- 2.3; ChR2 + BL: 51.6 +- 3.4). ** p < 0.01 vs. ChR2 determined by unpaired t -test. ( E ) Representative images of immunofluorescence staining for GFAP (red), nuclear factor-kappa B (NF-kB) (green), and nuclear detection by DAPI (blue) in unstimulated ChR2-NPCs (ChR2). Yellow arrows show nuclear staining. Scale bars, 1

Supportive validation

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Experimental details: Figure 2 AEG-1 knockdown reduces TWIK-1 protein levels and TWIK-1-mediated potassium currents in primary cultured astrocytes. ( A ) Immunofluorescence staining of cultured astrocytes using AEG-1 and GFAP, an astrocyte marker. ( B ) Immunofluorescence staining of TWIK-1 and AEG-1 in transfected astrocyte. Knockdown of the AEG-1 inhibits the levels of TWIK-1. Scale bar, 40 mum. The shRNA vector contains a shRNA sequence under the control of U6 promoter, with mCherry co-expression under a CMV promoter. ( C ) Knockdown of the AEG-1 in primary cultured astrocytes. Immunoblot data show the decreased AEG-1 and TWIK-1 expression. ( D ) Representative whole-cell I-V curves of astrocytes overexpressing Sc shRNA (black), AEG-1 shRNA (red), TWIK-1 shRNA (blue), and AEG-1 and TWIK-1 shRNAs (green). ( E ) Pooled data for whole-cell current amplitudes in AEG-1 knockdown, TWIK-1 knockdown, and AEG-1 and TWIK-1 double knockdown astrocytes. All values are presented as mean +- SEM. * p < 0.05, **** p < 0.0001. Two-way ANOVA followed by the Tukey's post hoc.

Supportive validation

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Experimental details: Figure 3 Neuroprotective effect of PA-C pre-treatment following peroxide-induced cytotoxicity in iPSC-NSC. ( A ). Effect of PA-C pre-treatment (5, 10, 12.5, 15, 17.5, and 20 uM) on iPSC-NSC viability following a 24 h incubation with 75 uM H 2 O 2 . MTS viability values represented as a percentage of vehicle-treated control cells. Data expressed as mean +- S.E.M. of three independent experiments determined by one-way ANOVA multiple comparison tests (*** p < 0.0001 vs. Control; ## p < 0.01 as indicated). ( B ). Representative bright-field images of iPSC-NSC morphology after PA-C pre-treatment and H 2 O 2 treatment (scale bar = 150 um). ( C ). Representative immunofluorescence staining of beta-III-Tubulin (green), GFAP (red), and DAPI (blue) of iPSC-NSC pre-treated for 30 min with 10 uM PA-C and then incubated with 75 uM H 2 O 2 for 24 h. Scale bar = 150 um. ( D ). Quantitative analysis of the ratio of beta-III-Tubulin cells and GFAP cells shown as a percentage of total DAPI from immunostaining. Data expressed as mean +- S.E.M. from three independent experiments as determined by one-way ANOVA with Tukey's multiple comparison test (*** p < 0.001 vs. Control; ### p < 0.001 as indicated).

Supportive validation

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Experimental details: Figure 6 ( A ). PA-C and PA-C combined with iPSC-NSC + MSC transplantation preserves beta-III-Tubulin fibers and synapses and reduces scar size A. Representative immunofluorescence images ( left panel ) of beta-III-Tubulin (green) and GFAP (red) of longitudinal spinal cord sections, including the injured area nine weeks after SCI (scale bar = 500 um). Dotted lines delimit the GFAP-negative area. ( B ). Quantification of beta-III-Tubulin-positive fibers ( upper panel ) and GFAP-negative scar area ( lower panel ) represented as a percentage of the total analyzed area and expressed as mean +- S.E.M. determined by one-way ANOVA with Tukey's multiple comparison test for beta-III-Tubulin and Kruskal-Wallis one-way ANOVA with Dunn's method for GFAP (control, n = 8; iPSC-NSC + MSC, n = 8; n = 4 for iPSC-NSC, MSC, PA-C, iPSC-NSC + MSC + PA-C). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. iPSC-NSC; && p < 0.01, &&& p < 0.001, vs. MSC, %%% p < 0.001 vs. iPSC-NSC + MSC, +++ p < 0.001 vs. PA-C. ( C ). Representative immunofluorescence images of longitudinal spinal cord sections of synaptophysin (green), NeuN (red), and DAPI (blue) staining (scale bar = 200 u). ( D ). Quantification of NeuN-positive area ( left panel ) and functional synapses by analyzing the co-localization of synaptophysin and NeuN ( right panel ), represented as a percentage of the NeuN-positive area. Quantitative data expressed as mean +- S.E.M determin

Supportive validation

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Experimental details: Fig. 3 Immunohistochemical analysis of the AEA system. A Immunohistochemistry of naive male and female cortex. Staining for NAPE-PLD, FAAH, Iba1, GFAP, and Nissl was performed. Original magnification x63. B Immunohistochemistry of naive male and female PAG. Staining for NAPE-PLD, FAAH, Iba1, GFAP, and Nissl was performed. Original magnification x63. Scale on lower left image applies to all frames. C Measurement of relative area of IHC field occupied by NAPE-PLD staining pixels for each sex and region. No significant differences were observed between region or sex. D Measurement of co-efficient of colocalization for NAPE-PLD with IBa1, Nissl, and GFAP. NAPE-PLD colocalized with Nissl at significantly higher rates in female cortex vs male cortex (**** p = 0.004). No significant differences observed in PAG. E Measurement of relative area of IHC field occupied by FAAH staining pixels for each sex and region. FAAH immunoreactivity was significantly higher in male cortex vs female cortex (**** p = 0.003). F Measurement of co-efficient of colocalization for FAAH with IBa1, Nissl, and GFAP. No significant differences observed between region or sex. All calculations for co-efficient of colocalization were performed with respect to the relevant enzyme. n = 3/sex for each analysis

Supportive validation

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Experimental details: Fig. 4 Immunohistochemical analysis of the AEA system. A Immunohistochemistry of naive male and female V1M cortex. Staining for NAPE-PLD, FAAH, Iba1, GFAP, and Nissl was performed. Original magnification x63. B Immunohistochemistry of naive male and female PAG. Staining for NAPE-PLD, FAAH, Iba1, GFAP, and Nissl was performed. Original magnification x63. C Measurement of relative area of IHC field occupied by NAPE-PLD staining pixels for each sex and region. Female NAPE-PLD immunoreactivity was significantly higher in PAG vs male PAG (* p = 0.02). D Measurement of co-efficient of colocalization for NAPE-PLD with IBa1, Nissl, and GFAP. NAPE-PLD colocalized with Nissl at significantly higher rates in female V1M cortex vs male V1M cortex (**** p = 0.001). No significant differences observed in PAG. E Measurement of relative area of IHC field occupied by FAAH staining pixels for each sex and region. FAAH immunoreactivity was significantly higher in female PAG vs male PAG (*** p = 0.0042) as well as female PAG vs female V1M cortex ( ### p = 0.002). F Measurement of co-efficient of colocalization for FAAH with IBa1, Nissl, and GFAP. No significant differences observed between region or sex. All calculations for co-efficient of colocalization were performed with respect to the relevant enzyme. n = 3/sex for each analysis

Supportive validation

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Experimental details: Fig. 5 Immunohistochemical analysis of CB1R and CB2R. A Immunohistochemistry of naive male and female cortex. Staining for CB1R, CB2R, Iba1, GFAP, and Nissl was performed. Original magnification x63. B Immunohistochemistry of naive male and female PAG. Staining for CB1R, CB2R, Iba1, GFAP, and Nissl was performed. Original magnification x63. Scale on lower left image applies to all frames. C Relative immunoreactivity of CB1R staining revealed significantly greater staining for CB1R in female cortex and lPAG vs males for both regions (**** p < 0.0001 female cortex vs male cortex, *** p = 0.001 female lPAG vs male lPAG). D Measurement of co-efficient of colocalization for CB1R with Iba1, Nissl, and GFAP. CB1R colocalization with Nissl was significantly higher in female cortex vs male cortex (*** p = 0.008) and female PAG vs male PAG (** p = 0.01). E CB2R immunoreactivity was significantly higher in female lPAG vs male lPAG (** p = 0.0053). F Measurement of co-efficient of colocalization for CB2R with IBa1, Nissl, and GFAP. CB2R colocalization with Nissl in the male cortex was significantly greater than in female cortex (*** p = 0.0178). All calculations for co-efficient of colocalization were performed with respect to the relevant enzyme. n = 3/sex for each analysis

Supportive validation

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Experimental details: Additional file 1: Figure 1. Regional analysis. A Serial sections from rats' brains were obtained at a thickness of 30um, moving posteriorly from approximately Bregma -7mm with PAG imaging in the lateral PAG. B Quantification of the relative area immunoreactive in each field for Nissl (neuronal soma), GFAP (astrocytes), and Iba1 (microglia) was statistically similar between regions and sex.

Supportive validation

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Experimental details: FIGURE 2 Confocal microscope images showing horizontal-sections of immuno-labeled cortical brain tissue along with cross sections of the perforated segment of an implanted PPMP, 2 weeks post implantation. Shown are: microglia (cyan) within and around the implant marked by asterisks (A) , astrocytes ( B , red), neurons and neurites ( C , green), and a merged image of (A-C) which also includes the nuclei of the cells labeled in yellow (D) . (E) Histograms depicting the average Normalized Fluorescent Intensity (NFI) or number of cells/100 mum 2 . Microglia ( E1 , cyan), astrocytes ( E2 , red), neurites and cell bodies ( E3 , green), and neuronal cell bodies ( E4 , green) within and around the platform's perforated segments. The time post-platform implantation is coded by the darkening of the column color as indicated by the legend on the right hand side of the histograms. The average NFI values or the cells/100 mum 2 within the platforms (PPMP) are highlighted in yellow. The distance of the average NFI from the MEA platform is given by shell number. Each shell is 25 mum wide (as illustrated in Figure 1F ). Vertical lines correspond to one standard deviation. The orange lines indicated by the arrowheads depict the normal NFI values or the number of cell/100 mum 2 in the control cortices. An enlarged image of (D) is presented as Supplementary Figure 2 .

Supportive validation

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Experimental details: Fig. 4 TLR2 and TLR4 loss influence the spontaneous differentiation of in vitro-expanded spinal cord NPCs. A , B Representative immunofluorescence images of beta3tubulin (neural marker; green) and GFAP (astrocytic marker, red) and the corresponding quantification data of the percentage of positive cells (lower panels) in NPCs grown in growth medium ( A ) or in differentiation medium ( B ) as summarized in the diagrams (top images). DAPI used for nuclei counterstaining (blue). C Morphological classification of the three distinct types of neurons identified from beta3tubulin staining--type 1 (pyramidal-like cells; upper panel), Type 2 (rounded, with no cell expansions; central panel), and Type 3 (bipolar cells; lower panel). Quantification and comparative analysis of the percentage of the corresponding type of neurons shown for WT, TLR2 -/- , and TLR4 -/- NPCs; D Gene expression analysis of Dcx (early neuronal marker, upper graph) and MAP2 (late neuronal marker, lower graph). E Gene expression analysis of Neurogenin1 in growth medium (left) or differentiation medium (right) in WT, TLR2 -/- and TLR4 -/- NPCs. F (lower graph) Quantification and comparative analysis versus WT NPCs of the number of cells expressing higher GFAP protein expression levels in TLR2 -/- and TLR4 -/- cells; (upper panels) Binarized representative images for TLR2 -/- and TLR4 -/- NPCs showing cells with a fluorescence intensity above the WT levels threshold. G Protein expression levels of STAT3 in WT, TLR2

Supportive validation

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Experimental details: Fluorescence staining of neuroglial culture for cell markers GFAP and beta-III tubulin. Fluorescent images of cell culture on days 3 ( A ) and 7 ( B ) after scratching (7 DIV and 10 DIV, respectively) and intact cell culture (10 DIV) without scratching ( C ) were taken with a fluorescent microscope Zeiss Axiovert-200, objective 40x/NA = 1.35 (oil). Scale bar is shown on the images. The yellow double arrow corresponds to the width of the scratch. Pairwise stitching ( A ) of two adjacent fragments was obtained using ImageJ software. ( A ) On day 3 after injury, a few neurons are observed in the injured area (indicated by numbers 1-3). ( B ) Numerous neurons migrate into the damaged area on day 7 after scratching.

Supportive validation

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Experimental details: Fluorescence staining of neuroglial culture for GFAP i beta-III tubulin. Fluorescent images of the cell culture were obtained on day 3 after scratching, 7 DIV ( A ); on day 7 after scratching, 10 DIV ( B ); on day 10 after scratching, 14 DIV ( C ), and intact cell culture without scratching, 14 DIV ( D ). Scale bar is shown on the images. The yellow double arrow corresponds to the width of the scratch. Images were acquired using a LSM 880 scanning laser confocal microscope equipped with an AiryScan module and GaAsP detector. The ZEN Black tile scanning function was used to stitch four separate images in each panel.

Supportive validation

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Experimental details: Constitutively active AKT signaling reduces E-cadherin expression via Snail transcriptional downregulation but does not affect DSG 2. (A) qRT - PCR analysis shows that overexpression of constitutively active AKT results in a 93% reduction in E-cadherin mRNA expression. (B) Western Blot analysis shows that E-cadherin protein expression is dramatically reduced in the MAH cell line. (C) Representative immunofluorescence (IF) analysis shows that E-cadherin is undetectable at the cell-cell border in MAH cell line. E-cadherin is shown in green, HA in red and DAPI in blue. (D) qRT - PCR analysis shows that DSG 2 mRNA expression is slightly increased (1.6X) in the MAH cell line. (E) Western Blot analysis confirms an increase in DSG 2 protein expression. (F) IF analysis shows that DSG 2 is detected at the cell-cell border in MAH cells, though less frequently than as compared to the DU 145 parental cell line by observation. Most of the cells that express DSG 2 at the cell border also express HA , although scattered cells with high HA expression displayed loss of DSG 2 (white arrows). DSG is shown in green, HA in red and DAPI in blue. (G) qRT - PCR analysis shows a slight but significant increase (1.8X) in Snail mRNA expression in the MAH cell line. (H) Western Blot analysis shows that Snail protein expression is comparable to that of the DU 145 parental cell line. (I) IF analysis shows a dramatic increase in the nuclear localization of Snail in the MAH cell line, indicative of Snail ac

Supportive validation

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Experimental details: Figure 2 3D immunofluorescence imaging of field RABV-infected neurons and astrocytes in a mouse brain. ( a ) Workflow for 3D immunofluorescence imaging, including vibratome sectioning into 1 mm slices, pretreatment, immunostaining, and subsequent optical clearing with organic solvents. Confocal imaging and acquisition of cleared tissue slices was done in custom-made imaging containers (see lower image). ( b , c ) Maximum z- and ( d - e ) 3D projections of z-stack (x, y, z = 400 um, 400 um, 59 um for D and 400 um, 400 um, 103 um for E) after indirect immunofluorescence for RABV phosphoprotein P (red), GFAP (green), and NeuN (blue). White arrows in Figure 2 b indicate RABV P and GFAP-positive astrocytes. ( f ) Maximum projection of detail from Figure 2 b (see white box) with GFAP-positive cell (green) and associated RABV P fluorescence (red). ( g ) 3D projection of detail view from Figure 2 f.

Supportive validation

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Experimental details: Figure 3 Quantification of RABV-infected neurons and astrocytes. ( a ) Maximum z-projection of objects map for NeuN-positive neurons generated from a confocal z-stack (see Figure 2 b). Individual neurons in the z-stack were identified by NeuN-specific fluorescence and converted to objects. Numbers indicate individual cell counts (for improved legibility of the individual numbers, refer to the enlarged details in Supplementary Video S3 ). n = 762 neurons in a volume of 400 um x 400 um x 59 um. The object colors indicate different z-positions (darker colors in the back and brighter colors in the front). ( b ) Detail of area indicated by white box in Figure 3 a. Green arrows indicate RABV-positive neuron cell bodies. ( c ) Overlay of objects map (greyscale) with RABV P signals allows identification and counting of rRABV Fox-infected neurons. ( d ) Maximum z-projection of objects map for GFAP-positive astrocytes generated from a confocal z-stack (see Figure 2 b). Individual astrocytes in the z-stack were identified by GFAP-specific fluorescence and converted to objects. Numbers indicate individual cell counts. n = 272 astrocytes in a volume of 400 um x 400 um x 59 um. The object colors indicate different z-positions (darker colors in the back and brighter colors in the front). ( e ) Detail of area indicated by white box in Figure 3 d. Green arrows indicate RABV-positive astrocytes. ( f ) Overlay of objects map (greyscale) with RABV P signals allows identification and counting of

Supportive validation

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Experimental details: Figure 4 Comparison of field and lab RABV-infected brains after intramuscular (i.m.) infection with rRABV Fox, rRABV Dog, rRABV Rac, rCVS-11, and Evelyn Rokitnicki Abelseth (ERA). ( a-e ) Maximum z-projections of z-stacks [x, y = 400 um, 400 um ( a-e ); z = 59 um ( a , rRABV Fox), 66 um ( b , rCVS-11), 49 um ( c , rRABV Dog), 100 um ( d , ERA) and 89 um ( e , rRABV Rac)] after indirect immunofluorescence for RABV phosphoprotein P (red), GFAP (green), and NeuN (blue). To improve visualization of the maximum z-projections, some z-stacks were reduced in thickness. For the full z-stacks, refer to Supplementary Video S4 . Insets in Figure 4 a,c,e show RABV P accumulation (red) at GFAP-positive cells (green). Insets in Figure 4 b,d show NeuN- (blue) and RABV P (red)-positive neurons. For the individual channels of the detail images, see Supplementary Figure S4 .

Supportive validation

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Experimental details: Figure 7 Comparison of field and lab RABV-infected brains after i.c. infection. ( a - f ) Maximum z-projections of z-stacks [x, y = 400 um, 400 um ( a - f ); z = 21 um ( a , rRABV Fox), 48 um ( b , rCVS-11), 74 um ( c , rRABV Dog), 98 um ( d , ERA), 75 um ( e , rRABV Rac) and 67 um ( f , SAD L16)] after indirect immunofluorescence for RABV phosphoprotein P (red), GFAP (green), and NeuN (blue). Insets in Figure 7 a,b,c,e show RABV P accumulation (red) at GFAP-positive cells (green). To improve visualization of the maximum z-projections, some z-stacks were reduced in thickness. Insets in Figure 7 d,f show NeuN- (blue) and RABV P (red)-positive neurons. For the individual channels of the detail images, see Supplementary Figure S5 .