goat anti p75  (R&D Systems)

Journal: eLife

Article Title: Lineage-specific intersection of endothelin and GDNF signaling in enteric nervous system development

doi: 10.7554/eLife.96424

Figure Lengend Snippet: ( a–d ) Wholemount preparations of E12.5 guts isolated from Pax2Cre/Ret/Rosa26 lacZ ( b ), Wnt1Cre/Ret/Rosa26 lacZ ( d ) mutant embryos and their littermate controls (a and c, respectively) stained with Xgal (blue). Arrows denote the wavefronts of lineage-labeled cells in the hindguts of the controls. Asterisks denote halted migration of Ret -deficient Pax2Cre -labeled cells at the cecum ( b ) and of Ret -deficient Wnt1Cre -labeled cells at the caudal end of the stomach ( d ). ( e–h ) Wholemount preparations of E12.5 guts isolated from Pax2Cre/Ret/Rosa26 td-Tomato ( f ), Wnt1Cre/Ret/Rosa26 td-Tomato ( h ) mutant embryos and their littermate controls (e and g, respectively) stained for neuronal 2H3 (green). Arrows denote the wavefronts of lineage-labeled cells in the hindguts of the control embryos. Arrowheads denote halted migration of Ret -deficient Pax2Cre -labeled cells at the cecum ( f ). ( i–l ) Wholemount preparations of E10.5 guts isolated from Pax2Cre/Ret/Rosa26 nT-nG ( j ), Wnt1Cre/Ret/Rosa26 nT-nG ( l ) mutant embryos and their littermate controls (i and k, respectively) stained for nuclear-GFP (green) and ENS progenitor marker p75 (magenta). Arrows denote the wavefronts of lineage-labeled cells in the midguts. Arrowheads denote p75 + Pax2Cre -labeled cells at the midgut areas ( i, j ). Abbreviations: es, esophagus; st, stomach. Scale bars, 500 μm ( a–d ), 250 μm ( e–h ), 50 μm ( i–l ).

Article Snippet: Antibodies used were rabbit anti-Pax2 (1:50, Invitrogen 08-1483), rabbit anti-AP2 (1:1000, Abcam ab52222), goat anti-Sox10 (1:200, Santa Cruz sc-17342), mouse anti-2H3 (1:100, DSHB), chicken anti-GFP (1:3000, Abcam ab13970), goat anti-p75 (1:300, R&D Systems AF1157), mouse anti-HuD (1:500, Santa Cruz sc-13577), rabbit anti-BLBP (1:500, Abcam ab32423), mouse anti-Tuj1 (1:1000, BioLegend MMS-435P), rabbit anti-CGRP (1:500-1000, Millipore PC205L), rabbit anti-NOS1 (1:500, Santa Cruz sc-648), goat anti-Ret (1:300, R&D Systems AF482), rabbit anti-phospho-Ret (Tyr1015) (1:300, Themo Fisher PA5-105930), and rabbit anti-phospho-Ret (Tyr1096) (1:300, Themo Fisher PA5-105796).

Techniques: Isolation, Mutagenesis, Staining, Labeling, Migration, Control, Marker

Journal: eLife

Article Title: Lineage-specific intersection of endothelin and GDNF signaling in enteric nervous system development

doi: 10.7554/eLife.96424

Figure Lengend Snippet: ( a–f ) Confocal z-stack images of 200 mm midgut sections isolated from E10.5 Pax2Cre/Rosa26 td-Tomato ( a–c ) and Wnt1Cre/Rosa26 td-Tomato embryos cultured for 3 days in the presence of GDNF ( a ), EDN3 ( b ), or NGF ( c ) and stained for neuronal Hu (cyan) and ENS progenitor marker p75 (yellow). Scale bar, 250 mm. ( g, h ) Compiled representations of the total area of outgrowth (Tomato + area around the explanted gut tube) from Pax2Cre/Rosa26 td-Tomato and Wnt1Cre/Rosa26 td-Tomato midgut slice explants in the presence of GDNF (black), EDN3 (orange), or NGF (blue). The data from GDNF-treated groups here are shown as the controls in . See for rostrocaudal orientation of these midgut slice preparations. Each dot represents one gut slice isolated from the indicated axial level; lost sections were not included as data points. The images shown in a–f represent sections that are 800–1000 μm caudal to the foregut-midgut junction (red boxes in g and h). p-Values: *p<0.05, **p<0.01, ***p<0.001, ns = not significant.

Article Snippet: Antibodies used were rabbit anti-Pax2 (1:50, Invitrogen 08-1483), rabbit anti-AP2 (1:1000, Abcam ab52222), goat anti-Sox10 (1:200, Santa Cruz sc-17342), mouse anti-2H3 (1:100, DSHB), chicken anti-GFP (1:3000, Abcam ab13970), goat anti-p75 (1:300, R&D Systems AF1157), mouse anti-HuD (1:500, Santa Cruz sc-13577), rabbit anti-BLBP (1:500, Abcam ab32423), mouse anti-Tuj1 (1:1000, BioLegend MMS-435P), rabbit anti-CGRP (1:500-1000, Millipore PC205L), rabbit anti-NOS1 (1:500, Santa Cruz sc-648), goat anti-Ret (1:300, R&D Systems AF482), rabbit anti-phospho-Ret (Tyr1015) (1:300, Themo Fisher PA5-105930), and rabbit anti-phospho-Ret (Tyr1096) (1:300, Themo Fisher PA5-105796).

Techniques: Isolation, Cell Culture, Staining, Marker

Journal: bioRxiv

Article Title: Cholinergic basal forebrain neurons regulate vascular dynamics and cerebrospinal fluid flux

doi: 10.1101/2024.08.25.609536

Figure Lengend Snippet: a) An immunotoxin, p75-saporin, which targets the p75 neurotrophin receptor-expressing cholinergic neurons was injected into the medial septum (MS) of the mouse basal forebrain. This resulted in significant reduction of cholinergic neurons (green fluorescence) and their density in the MS compared to that of sham mice injected with the non-targeted IgG-saporin. b) 3-4 weeks after the surgery, resting-state fMRI was conducted using a 9.4T MRI to measure the cortical BOLD (dash line) and ventricular CSF inflow (solid line) signals. c) The cross-correlation between the cortical BOLD and CSF signals shows anticorrelation at 0 lag time in both sham and lesioned mice. However, the anticorrelation between the regional BOLD and CSF signals were abolished in the lesioned mice in the hippocampus, which receives the MS cholinergic projection, but not in the cortex. Error bars represent the SEM. d) The cortical BOLD signal coupling with the CSF signal (top) and fluctuation amplitude (bottom) were comparable between the lesioned and sham mice. e) In the hippocampus, however, the hippocampal BOLD-CSF coupling strength was reduced toward 0, with the BOLD signal amplitude showed a trend of decrease. f) The hippocampal BOLD-CSF coupling, BOLD signal amplitude, and the lag time of maximal anticorrelation between the BOLD and CSF signals, were all significantly correlated with the p75-positive cholinergic neuron density of the MS of the imaged mice.

Article Snippet: Immunofluorescence labeling of the cholinergic cells were stained with primary goat anti-p75 antibody (1:400, AF1157, R&D Systems) overnight at room temperature.

Techniques: Expressing, Injection, Fluorescence

Journal: Cells

Article Title: Etiological Roles of p75 NTR in a Mouse Model of Wet Age-Related Macular Degeneration

doi: 10.3390/cells12020297

Figure Lengend Snippet: MPCs expressing p75 NTR migrate into the laser-injured area of RPE-Choroid. p75 NTR co-localizes in F4/80 positive cells in RPE-Choroid. Tissue extracts and flat-mounted retinas were prepared and evaluated by Western blot and by IHC. ( A ) Representative Western blot of RPE-Choroid homogenates prepared from WT mice without CNV, or 4 days after laser injury. Tubulin was used as loading control. Bands were quantified by densitometric analysis, and p75 NTR /tubulin ratio is represented in the bar graph expressed as units relative to control. Bars denote the mean ± SD from triplicate experiments, n = 5 mice/group. The asterisks show statistical differences respect to control. * p < 0.05. ( B ) Representative Western blot of RPE-Choroid homogenates prepared from WT mice without CNV, or 7 days after laser injury. Tubulin was used as loading control. Bands were quantified by densitometric analysis, and p75 NTR /tubulin ratio is represented in the bar graph expressed as units relative to control. ns: non-significant. Bars denote the mean ± SD from triplicate experiments, n = at least 4 mice/group. ( C – E ) Representative confocal images of RPE-Choroid flat-mounts of WT mice 4 days after laser injury, showing immunofluorescence staining with: ( C ) F4/80 (green), p75 NTR (red) and cell nuclei (blue). Scale bar upper panel: 50 µm. Scale bar lower panel: 20 µm. ( D ) IBA-1 (green), p75 NTR (red) and cell nuclei (blue). Scale bar upper panel: 100 µm. Scale bar lower panel: 50 µm. ( E ) CX3CR1 (green), p75 NTR (red) and cell nuclei (blue). Scale bar upper panel: 100 µm. Scale bar lower panel: 25 µm. ( F , G ) Representative confocal images of RPE-Choroid flat-mounts of WT mice 7 days after laser injury, showing immunofluorescence staining with ( F ) Isolectin IB-4 (green), p75 NTR (red) and cell nuclei (blue). Scale bar: 50 µm. ( G ) NG-2 (green), p75 NTR (red) and cell nuclei (blue). Scale bar: 50 µm.

Article Snippet: Afterwards, RPE-Choroids were incubated overnight with 0.01 μg/μL of Isolectin IB4 Alexa fluor-488 conjugate (GSA-IB4) from Molecular Probes, Inc. (Eugene, OR, USA), phalloidin-Alexa Fluor 647 (Thermo Fisher Scientific, A22287, Waltham, MA, USA), goat polyclonal anti-p75 NTR (1/500; R&D system), mouse anti f4/80 (1/50; Invitrogen, Waltham, MA, USA), rabbit anti CX3CR1 (1/100; Abcam, ab8021, Cambridge, UK), rabbit polyclonal anti-iba1 (1/200; Wako 019-19741), or rabbit polyclonal anti-NG-2 (1/100; AB5320, Millipore, Burlington, MA, USA).

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Staining

Journal: Cells

Article Title: Etiological Roles of p75 NTR in a Mouse Model of Wet Age-Related Macular Degeneration

doi: 10.3390/cells12020297

Figure Lengend Snippet: Activated macroglial cells express p75 NTR after the laser around the injured area in the retina. p75 NTR protein co-localizes with GFAP-positive activated macroglia in CNV mice retinas, 7 days after laser. Tissue extracts and retina sections were prepared and evaluated by Western blot and by IHC. ( A ) Representative Western blot of total retinas homogenates prepared from WT mice without CNV, or 7 days after laser injury. (+) Correspond to a positive control (hippocampus E19 M2). Actin was used as loading control. Bands were quantified by densitometric analysis, and p75 NTR /actin ratio is represented in the bar graph expressed as units relative to control. Bars denote the mean ± SD from triplicate experiments, n = 6 mice/group. The asterisks show statistical differences respect to control. * p < 0.05. ( B ) Representative confocal images of WT CNV mice retinal cryosections, 7 days after laser. Upper panel: immunofluorescence staining of GFAP (green) and p75 NTR (red). Scale bar: 50 µm. * Indicates the injured area. Zoom scale bar: 25 µm. Cell nuclei counterstained with Hoechst are also shown (blue). Lower panel: immunofluorescence staining of β3 tubulin (green) and p75 NTR (red). Scale bar: 25 µm. Zoom scale bar: 10 µm. Abbreviations: GCL (ganglion cells layer), IPL (inner plexiform layer), INL (inner nuclear layer), OPL (outer plexiform layer), ONL (outer nuclear layer). ( C ) Representative confocal images of retinal flat-mount of WT CNV mice, 7 days after laser, showing immunofluorescence staining with NG-2 (green) and p75 NTR (red). Scale bar: 50 µm.

Article Snippet: Afterwards, RPE-Choroids were incubated overnight with 0.01 μg/μL of Isolectin IB4 Alexa fluor-488 conjugate (GSA-IB4) from Molecular Probes, Inc. (Eugene, OR, USA), phalloidin-Alexa Fluor 647 (Thermo Fisher Scientific, A22287, Waltham, MA, USA), goat polyclonal anti-p75 NTR (1/500; R&D system), mouse anti f4/80 (1/50; Invitrogen, Waltham, MA, USA), rabbit anti CX3CR1 (1/100; Abcam, ab8021, Cambridge, UK), rabbit polyclonal anti-iba1 (1/200; Wako 019-19741), or rabbit polyclonal anti-NG-2 (1/100; AB5320, Millipore, Burlington, MA, USA).

Techniques: Western Blot, Positive Control, Control, Immunofluorescence, Staining

Journal: Cells

Article Title: Etiological Roles of p75 NTR in a Mouse Model of Wet Age-Related Macular Degeneration

doi: 10.3390/cells12020297

Figure Lengend Snippet: Reduced inflammatory phenotype in the RPE-Choroids and retinas of p75 NTR knockout mice, after laser injury. Mononuclear phagocytic cell recruitment is significantly reduced in retinas and RPE-Choroids of p75 NTR KO mice after CNV. ( A ) Representative flow cytometry pseudocolor plots from WT and p75 NTR KO mice without CNV, or 4 days after laser injury. Cells in the gate were quantified and the number of cells in the gate/total cells ratio is represented in the bar graph expressed as units relative to WT no laser control. Graphs denote the mean ± SD from triplicate experiments, n = 6 mice/group. The asterisks show statistical differences respect to control. * p < 0.05, ** p < 0.01, *** p < 0.001. ( B ) Representative confocal images of RPE-Choroid flat-mounts of WT and p75 NTR KO mice 4 days after laser, showing immunofluorescence staining with Isolectin IB-4 (grey) and F4/80 (green). Scale bar: 200 µm. F4/80 fluorescence intensity and area were quantified with ImageJ FIJI software Version 1.53t, and represented in the bar graph expressed as units relative to CNV WT control. Bars denote the mean ± SD from triplicate experiments, n = at least 5 mice/group. ns: non-significant.

Article Snippet: Afterwards, RPE-Choroids were incubated overnight with 0.01 μg/μL of Isolectin IB4 Alexa fluor-488 conjugate (GSA-IB4) from Molecular Probes, Inc. (Eugene, OR, USA), phalloidin-Alexa Fluor 647 (Thermo Fisher Scientific, A22287, Waltham, MA, USA), goat polyclonal anti-p75 NTR (1/500; R&D system), mouse anti f4/80 (1/50; Invitrogen, Waltham, MA, USA), rabbit anti CX3CR1 (1/100; Abcam, ab8021, Cambridge, UK), rabbit polyclonal anti-iba1 (1/200; Wako 019-19741), or rabbit polyclonal anti-NG-2 (1/100; AB5320, Millipore, Burlington, MA, USA).

Techniques: Knock-Out, Flow Cytometry, Control, Immunofluorescence, Staining, Fluorescence, Software

Journal: Cells

Article Title: Etiological Roles of p75 NTR in a Mouse Model of Wet Age-Related Macular Degeneration

doi: 10.3390/cells12020297

Figure Lengend Snippet: Reduced neovascular phenotype in the choroid of p75 NTR knockout mice, after laser injury. The area and the perimeter of choroidal neovessels are significantly reduced in RPE-Choroid flat-mounts of p75 NTR KO mice. ( A ) Representative confocal images of RPE-Choroid flat-mounts of WT and p75 NTR KO mice 1 day after laser injury, showing immunofluorescence staining with Falloidin (green). The yellow outline represents the lesioned area estimated by F-actin negative staining. Scale bar: 15 µm. Cell nuclei counterstained with Hoechst are also shown (blue). The area and perimeter of the laser lesion were quantified with ImageJ FIJI software, and represented in the bar graph expressed as units relative to WT CNV control. Bars denote the mean ± SD from triplicate experiments, n = 4 mice/group. ns: non-significant. ( B ) Representative confocal images of RPE-Choroid flat-mounts of WT mice 7 days after laser injury, showing immunofluorescence staining with Isolectin IB-4 (green). The yellow outline represents the neovessels covered area. Scale bar: 200 µm. The area and the perimeter of the neovessels were quantified with ImageJ FIJI software, and represented in the bar graph expressed as units relative to CNV WT control. Bars denote the mean ± SD from triplicate experiments, n = at least 6 mice/group. The asterisks show statistical differences respect to control. * p < 0.05, ** p < 0.01, *** p < 0.001. ( C ) Representative Western blot of total retinal homogenates prepared from WT and p75 NTR KO mice without CNV, or 7 days after laser injury. Tubulin was used as loading control. Bands were quantified by densitometric analysis, and GFAP/tubulin ratio is represented in the bar graph expressed as units relative to control. Bars denote the mean ± SD from triplicate experiments, n = 3 mice/group. ( D ) Representative immunofluorescence analysis of GFAP (green) in retinal cryosections from WT and p75 NTR KO mice without injury or 7 days after laser injury. Scale bar: 50 µm. Cell nuclei counterstained with Hoechst are also shown (blue). Abbreviations: GCL (ganglion cells layer), IPL (inner plexiform layer), INL (inner nuclear layer), OPL (outer plexiform layer), ONL (outer nuclear layer). ( E ) Amplitudes and implicit times of a- and b-waves from scotopic electroretinograms recorded in WT and p75 NTR KO mice without injury or 7 days after laser injury. The data show averages of responses of both eyes. Graphs denote the mean ± SD from triplicate experiments, n = at least 6 mice/group. The symbol correspond: WT no laser (filled circle), WT CNV (filled square), p75 NTR KO no laser (filled triangle up), p75 NTR KO CNV (filled triangle down). The asterisks show statistical differences respect to control. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Afterwards, RPE-Choroids were incubated overnight with 0.01 μg/μL of Isolectin IB4 Alexa fluor-488 conjugate (GSA-IB4) from Molecular Probes, Inc. (Eugene, OR, USA), phalloidin-Alexa Fluor 647 (Thermo Fisher Scientific, A22287, Waltham, MA, USA), goat polyclonal anti-p75 NTR (1/500; R&D system), mouse anti f4/80 (1/50; Invitrogen, Waltham, MA, USA), rabbit anti CX3CR1 (1/100; Abcam, ab8021, Cambridge, UK), rabbit polyclonal anti-iba1 (1/200; Wako 019-19741), or rabbit polyclonal anti-NG-2 (1/100; AB5320, Millipore, Burlington, MA, USA).

Techniques: Knock-Out, Immunofluorescence, Staining, Negative Staining, Software, Control, Western Blot

Journal: Cells

Article Title: Etiological Roles of p75 NTR in a Mouse Model of Wet Age-Related Macular Degeneration

doi: 10.3390/cells12020297

Figure Lengend Snippet: Reduced neovascular phenotype and improved retinal function in p75 NTR antagonist-treated wild type mice after laser injury. p75 NTR receptor antagonist reduced the area and perimeter of choroidal neovessels and improved retinal functionality in WT CNV mice. ( A ) Representative confocal images of RPE-Choroid flat-mounts of WT mice 7 days after laser, showing immunofluorescence staining with Isolectin IB-4 (grey). Scale bar: 100 µm. ( B ) The area and perimeter of neovessels were quantified with ImageJ FIJI software and represented in the bar graph expressed as units relative to WT CNV vehicle control. Bars denote the mean ± SD from triplicate experiments, n = 5 mice/group. ( C ) Amplitudes and implicit times of a- and b-waves from scotopic electroretinograms recorded 7 days after the laser in WT and WT CNV mice injected with THX-B or vehicle. The data show averages of responses of both. Graphs denote the mean ± SD from triplicate experiments, n = at least 6 mice/group. ( D ) Representative flow cytometry pseudocolor plots from WT CNV and no laser mice injected with THX-B or vehicle, 4 days after laser. Cells in the gate were quantified and the number of cells in the gate/total cells ratio is represented in the bar graph expressed as units relative to WT no laser vehicle control. Graphs denote the mean ± SD from triplicate experiments, n = 6 mice/group. The symbol correspond: No laser (white circle), CNV Vehicle (filled square), No laser THX B (filled triangle up), CNV THX B (filled triangle down). The asterisks show statistical differences respect to control. * p < 0.05, ** p < 0.01, *** p < 0.001. ( E ) Representative confocal images of RPE-Choroid flat-mounts of WT mice 4 days after laser, showing immunofluorescence staining with F4/80 (green). Scale bar: 200 µm. The area and the F4/80 fluorescence intensity were quantified with ImageJ FIJI software and represented in the bar graph expressed as units relative to WT CNV+ vehicle control. Bars denote the mean ± SD from triplicate experiments, n = 4 mice/group. ns: non-significant.

Article Snippet: Afterwards, RPE-Choroids were incubated overnight with 0.01 μg/μL of Isolectin IB4 Alexa fluor-488 conjugate (GSA-IB4) from Molecular Probes, Inc. (Eugene, OR, USA), phalloidin-Alexa Fluor 647 (Thermo Fisher Scientific, A22287, Waltham, MA, USA), goat polyclonal anti-p75 NTR (1/500; R&D system), mouse anti f4/80 (1/50; Invitrogen, Waltham, MA, USA), rabbit anti CX3CR1 (1/100; Abcam, ab8021, Cambridge, UK), rabbit polyclonal anti-iba1 (1/200; Wako 019-19741), or rabbit polyclonal anti-NG-2 (1/100; AB5320, Millipore, Burlington, MA, USA).

Techniques: Immunofluorescence, Staining, Software, Control, Injection, Flow Cytometry, Fluorescence

Journal: Molecular Vision

Article Title: Nerve growth factor modulates toll-like receptor (TLR) 4 and 9 expression in cultured primary VKC conjunctival epithelial cells

doi:

Figure Lengend Snippet: TrkA NGFR /p75 NTR protein expression in primary untouched VKC conjunctival epithelial cells (VKC-ECs). Confocal analysis specific for trkA NGFR (middle) and p75 NTR (right) in VKC-ECs. Control-isotype signal (left) was used in channel series acquisitions, and identical acquisition settings were applied for all images (60× oil immersion).

Article Snippet: A brief blocking/permeabilizing step (0.8% bovine serum albumin [BSA] and 0.3% Triton-X100 in PBS) was performed before the addition of the specific antibodies: rabbit anti-human TLR4 (sc-10741; Santa Cruz Biotech, Santa Cruz, CA), goat anti-human TLR9 (sc-16247; Santa Cruz Biotech), mouse anti-human trkA NGFR and goat anti-human p75 NTR antibodies (0.5–1 µg/ml; R&D).

Techniques: Expressing