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mouse anti βiii tubulin  (R&D Systems)


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    R&D Systems mouse anti βiii tubulin
    (A) Schematic of the neuron-astrocyte coculture . (B) Representative images of the neuron-astrocyte coculture. Coculture of GFP-LC3 transgenic astrocytes and non-transgenic neurons were fixed on DIV7 of coculture (neurons were a total of DIV11) and immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), AQP4 (astrocyte marker) and <t>β3-tubulin</t> (neuron marker); nuclei were labeled with Hoechst (shown in blue in the merged image). Shown are maximum projections of z-stacks. Dashed box indicates location of the zoom-in. Scale bars, 10 µm. (C) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and LAMP1. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for GFP(LC3), p62, and LAMP1 are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate puncta co-positive for GFP(LC3) and LAMP1, or p62 and LAMP1. Empty yellow arrowheads indicate GFP(LC3) or p62 puncta that are negative for LAMP1. Scale bars, 10 µm. ( C’ ) Representative line scans of p62 and LAMP1-positive puncta in the soma of neurons and astrocytes. Asterisks denote peaks co-positive for p62 and LAMP1. (D) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and ubiquitin; nuclei (Nuc) were labeled with Hoechst. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for p62 and ubiquitin are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate colocalization between p62 puncta and ubiquitin puncta. Empty yellow arrowheads indicate p62 puncta with no ubiquitin puncta correlate. Scale bar, 10 µm. (E-E’) Quantification of the total area occupied by p62-positive puncta normalized to soma area for neurons (E) or astrocytes (E’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 37-40 neurons and N = 27-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (F-F’) Quantification of the total area occupied by ubiquitin-positive puncta normalized to soma area for neurons (F) or astrocytes (F’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 35-36 neurons and N = 29-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (G-G’) Quantification of the percentage of overlapping area between p62-positive puncta and ubiquitin-positive puncta normalized to soma area for neurons (G) or astrocytes (G’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 32-33 neurons and N = 23-30 astrocytes from 3 independent experiments; 7-8 DIV of coculture. Throughout the figure, neurons are circled in orange and astrocytes are circled in purple. For all graphs in all figures, small circles indicate the measurements from individual cells (e.g., the technical replicates) from each of the independent experiments; large triangles indicate the corresponding biological means from each of the independent experiments (e.g., the biological replicates); independent experiments are color-coded.
    Mouse Anti βiii Tubulin, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 625 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+anti+%CE%B2+iii+tubulin/Neuron-specific+beta-III+Tubulin+Antibody/pmc13095109-31-5-8
    Average 96 stars, based on 625 article reviews
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    Images

    1) Product Images from "Differential regulation of p62-ubiquitin conjugates in neurons versus astrocytes during cellular stress"

    Article Title: Differential regulation of p62-ubiquitin conjugates in neurons versus astrocytes during cellular stress

    Journal: PLOS One

    doi: 10.1371/journal.pone.0345890

    (A) Schematic of the neuron-astrocyte coculture . (B) Representative images of the neuron-astrocyte coculture. Coculture of GFP-LC3 transgenic astrocytes and non-transgenic neurons were fixed on DIV7 of coculture (neurons were a total of DIV11) and immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), AQP4 (astrocyte marker) and β3-tubulin (neuron marker); nuclei were labeled with Hoechst (shown in blue in the merged image). Shown are maximum projections of z-stacks. Dashed box indicates location of the zoom-in. Scale bars, 10 µm. (C) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and LAMP1. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for GFP(LC3), p62, and LAMP1 are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate puncta co-positive for GFP(LC3) and LAMP1, or p62 and LAMP1. Empty yellow arrowheads indicate GFP(LC3) or p62 puncta that are negative for LAMP1. Scale bars, 10 µm. ( C’ ) Representative line scans of p62 and LAMP1-positive puncta in the soma of neurons and astrocytes. Asterisks denote peaks co-positive for p62 and LAMP1. (D) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and ubiquitin; nuclei (Nuc) were labeled with Hoechst. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for p62 and ubiquitin are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate colocalization between p62 puncta and ubiquitin puncta. Empty yellow arrowheads indicate p62 puncta with no ubiquitin puncta correlate. Scale bar, 10 µm. (E-E’) Quantification of the total area occupied by p62-positive puncta normalized to soma area for neurons (E) or astrocytes (E’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 37-40 neurons and N = 27-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (F-F’) Quantification of the total area occupied by ubiquitin-positive puncta normalized to soma area for neurons (F) or astrocytes (F’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 35-36 neurons and N = 29-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (G-G’) Quantification of the percentage of overlapping area between p62-positive puncta and ubiquitin-positive puncta normalized to soma area for neurons (G) or astrocytes (G’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 32-33 neurons and N = 23-30 astrocytes from 3 independent experiments; 7-8 DIV of coculture. Throughout the figure, neurons are circled in orange and astrocytes are circled in purple. For all graphs in all figures, small circles indicate the measurements from individual cells (e.g., the technical replicates) from each of the independent experiments; large triangles indicate the corresponding biological means from each of the independent experiments (e.g., the biological replicates); independent experiments are color-coded.
    Figure Legend Snippet: (A) Schematic of the neuron-astrocyte coculture . (B) Representative images of the neuron-astrocyte coculture. Coculture of GFP-LC3 transgenic astrocytes and non-transgenic neurons were fixed on DIV7 of coculture (neurons were a total of DIV11) and immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), AQP4 (astrocyte marker) and β3-tubulin (neuron marker); nuclei were labeled with Hoechst (shown in blue in the merged image). Shown are maximum projections of z-stacks. Dashed box indicates location of the zoom-in. Scale bars, 10 µm. (C) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and LAMP1. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for GFP(LC3), p62, and LAMP1 are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate puncta co-positive for GFP(LC3) and LAMP1, or p62 and LAMP1. Empty yellow arrowheads indicate GFP(LC3) or p62 puncta that are negative for LAMP1. Scale bars, 10 µm. ( C’ ) Representative line scans of p62 and LAMP1-positive puncta in the soma of neurons and astrocytes. Asterisks denote peaks co-positive for p62 and LAMP1. (D) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and ubiquitin; nuclei (Nuc) were labeled with Hoechst. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for p62 and ubiquitin are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate colocalization between p62 puncta and ubiquitin puncta. Empty yellow arrowheads indicate p62 puncta with no ubiquitin puncta correlate. Scale bar, 10 µm. (E-E’) Quantification of the total area occupied by p62-positive puncta normalized to soma area for neurons (E) or astrocytes (E’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 37-40 neurons and N = 27-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (F-F’) Quantification of the total area occupied by ubiquitin-positive puncta normalized to soma area for neurons (F) or astrocytes (F’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 35-36 neurons and N = 29-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (G-G’) Quantification of the percentage of overlapping area between p62-positive puncta and ubiquitin-positive puncta normalized to soma area for neurons (G) or astrocytes (G’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 32-33 neurons and N = 23-30 astrocytes from 3 independent experiments; 7-8 DIV of coculture. Throughout the figure, neurons are circled in orange and astrocytes are circled in purple. For all graphs in all figures, small circles indicate the measurements from individual cells (e.g., the technical replicates) from each of the independent experiments; large triangles indicate the corresponding biological means from each of the independent experiments (e.g., the biological replicates); independent experiments are color-coded.

    Techniques Used: Transgenic Assay, Marker, Labeling, Solvent, Control, Ubiquitin Proteomics

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    Incubation:

    Article Title: AraC binds the p75 NTR transmembrane domain to induce neurodegeneration in mature neurons
    Article Snippet: .. Cells were incubated overnight at 4 °C with mouse anti-β-III tubulin (R&D Systems, MAB1195; 1:2500) and counterstained the following day with donkey anti-mouse Alexa Fluor 488 (Abcam, ab150105; 1:2000) and Hoechst (Sigma, B2261; 1:2000). .. Images were taken using a Confocal Microscope LSM 780 Zeiss Axio Observer fluorescence microscope.

    Article Title: AraC interacts with p75 NTR transmembrane domain to induce cell death of mature neurons.
    Article Snippet: Cells were then permeabilized and blocked in 5% normal donkey serum (Jackson ImmunoResearch: 017-000- 121) and 0.3% Triton X-100 (Thermo Scientific; 85111) in PBS. .. Cells were incubated overnight at 4 °C with mouse anti-β-III tubulin (R&D Systems, MAB1195; 1:2500) and counterstained the following day with donkey antimouse Alexa Fluor 488 (Abcam, ab150105; 1:2000) and Hoechst (Sigma, B2261; 1:2000). .. Images were taken using a Confocal Microscope LSM 780 Zeiss Axio Observer fluorescence microscope.

    Article Title: AraC interacts with p75 NTR transmembrane domain to induce cell death of mature neurons
    Article Snippet: Cells were then permeabilized and blocked in 5% normal donkey serum (Jackson ImmunoResearch: 017-000-121) and 0.3% Triton X-100 (Thermo Scientific; 85111) in PBS. .. Cells were incubated overnight at 4 °C with mouse anti-β-III tubulin (R&D Systems, MAB1195; 1:2500) and counterstained the following day with donkey anti-mouse Alexa Fluor 488 (Abcam, ab150105; 1:2000) and Hoechst (Sigma, B2261; 1:2000). .. Images were taken using a Confocal Microscope LSM 780 Zeiss Axio Observer fluorescence microscope.

    Staining:

    Article Title: A novel method for generating glutamatergic SH-SY5Y neuron-like cells utilizing B-27 supplement.
    Article Snippet: .. Cells were fixed with 4% PFA for 20 min. Microtubules were stained with 1:500 mouse anti-β(III)-Tubulin (R&D Systems, #MAB1195) and visualized with 1:400 Alexa Fluor® 488-conjugated donkey anti-mouse (Thermo Scientific, #A21202) (green). ..

    Article Title: Lrp1 facilitates infection of neurons by Jamestown Canyon virus
    Article Snippet: .. The following antibodies were used for immunofluorescent staining during this study: mouse anti-β III-tubulin (1:500; R&D Systems, MAB1195), custom rabbit anti-JCV N (1:200; Genscript, Y743THG190-16), rabbit anti-Lrp1 (1:500; Abcam, ab92544), antisera from mice immunized with a sublethal dose of JCV (1:200; generated in house), goat anti-rabbit 488 (1:500; Invitrogen, A11008), goat anti-mouse 488 (1:500; Invitrogen, A11001), goat anti-rabbit 594 (1:500; Invitrogen, A11012), and goat anti-mouse 594 (1:500; Invitrogen, A11005). .. Cells were inactivated in 100μl of radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, 89901) with 1% Halt Protease Inhibitor (Thermo Fisher Scientific, 78429) for 10 min at room temperature.

    Article Title: A novel method for generating glutamatergic SH-SY5Y neuron-like cells utilizing B-27 supplement.
    Article Snippet: .. Cells were fixed with 4% PFA for 20 min. Microtubules were stained with 1: 500 mouse anti-β(III)-Tubulin (R&D Systems, #MAB1195) and visualized with 1:400 Alexa Fluor® 488-conjugated donkey anti-mouse (Thermo Scientific, #A21202) (green). ..

    Generated:

    Article Title: Lrp1 facilitates infection of neurons by Jamestown Canyon virus
    Article Snippet: .. The following antibodies were used for immunofluorescent staining during this study: mouse anti-β III-tubulin (1:500; R&D Systems, MAB1195), custom rabbit anti-JCV N (1:200; Genscript, Y743THG190-16), rabbit anti-Lrp1 (1:500; Abcam, ab92544), antisera from mice immunized with a sublethal dose of JCV (1:200; generated in house), goat anti-rabbit 488 (1:500; Invitrogen, A11008), goat anti-mouse 488 (1:500; Invitrogen, A11001), goat anti-rabbit 594 (1:500; Invitrogen, A11012), and goat anti-mouse 594 (1:500; Invitrogen, A11005). .. Cells were inactivated in 100μl of radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, 89901) with 1% Halt Protease Inhibitor (Thermo Fisher Scientific, 78429) for 10 min at room temperature.



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    (A) Schematic of the neuron-astrocyte coculture . (B) Representative images of the neuron-astrocyte coculture. Coculture of GFP-LC3 transgenic astrocytes and non-transgenic neurons were fixed on DIV7 of coculture (neurons were a total of DIV11) and immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), AQP4 (astrocyte marker) and <t>β3-tubulin</t> (neuron marker); nuclei were labeled with Hoechst (shown in blue in the merged image). Shown are maximum projections of z-stacks. Dashed box indicates location of the zoom-in. Scale bars, 10 µm. (C) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and LAMP1. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for GFP(LC3), p62, and LAMP1 are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate puncta co-positive for GFP(LC3) and LAMP1, or p62 and LAMP1. Empty yellow arrowheads indicate GFP(LC3) or p62 puncta that are negative for LAMP1. Scale bars, 10 µm. ( C’ ) Representative line scans of p62 and LAMP1-positive puncta in the soma of neurons and astrocytes. Asterisks denote peaks co-positive for p62 and LAMP1. (D) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and ubiquitin; nuclei (Nuc) were labeled with Hoechst. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for p62 and ubiquitin are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate colocalization between p62 puncta and ubiquitin puncta. Empty yellow arrowheads indicate p62 puncta with no ubiquitin puncta correlate. Scale bar, 10 µm. (E-E’) Quantification of the total area occupied by p62-positive puncta normalized to soma area for neurons (E) or astrocytes (E’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 37-40 neurons and N = 27-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (F-F’) Quantification of the total area occupied by ubiquitin-positive puncta normalized to soma area for neurons (F) or astrocytes (F’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 35-36 neurons and N = 29-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (G-G’) Quantification of the percentage of overlapping area between p62-positive puncta and ubiquitin-positive puncta normalized to soma area for neurons (G) or astrocytes (G’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 32-33 neurons and N = 23-30 astrocytes from 3 independent experiments; 7-8 DIV of coculture. Throughout the figure, neurons are circled in orange and astrocytes are circled in purple. For all graphs in all figures, small circles indicate the measurements from individual cells (e.g., the technical replicates) from each of the independent experiments; large triangles indicate the corresponding biological means from each of the independent experiments (e.g., the biological replicates); independent experiments are color-coded.
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    R&D Systems mouse monoclonal anti βiii tubulin
    (A) Schematic of the neuron-astrocyte coculture . (B) Representative images of the neuron-astrocyte coculture. Coculture of GFP-LC3 transgenic astrocytes and non-transgenic neurons were fixed on DIV7 of coculture (neurons were a total of DIV11) and immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), AQP4 (astrocyte marker) and <t>β3-tubulin</t> (neuron marker); nuclei were labeled with Hoechst (shown in blue in the merged image). Shown are maximum projections of z-stacks. Dashed box indicates location of the zoom-in. Scale bars, 10 µm. (C) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and LAMP1. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for GFP(LC3), p62, and LAMP1 are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate puncta co-positive for GFP(LC3) and LAMP1, or p62 and LAMP1. Empty yellow arrowheads indicate GFP(LC3) or p62 puncta that are negative for LAMP1. Scale bars, 10 µm. ( C’ ) Representative line scans of p62 and LAMP1-positive puncta in the soma of neurons and astrocytes. Asterisks denote peaks co-positive for p62 and LAMP1. (D) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and ubiquitin; nuclei (Nuc) were labeled with Hoechst. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for p62 and ubiquitin are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate colocalization between p62 puncta and ubiquitin puncta. Empty yellow arrowheads indicate p62 puncta with no ubiquitin puncta correlate. Scale bar, 10 µm. (E-E’) Quantification of the total area occupied by p62-positive puncta normalized to soma area for neurons (E) or astrocytes (E’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 37-40 neurons and N = 27-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (F-F’) Quantification of the total area occupied by ubiquitin-positive puncta normalized to soma area for neurons (F) or astrocytes (F’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 35-36 neurons and N = 29-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (G-G’) Quantification of the percentage of overlapping area between p62-positive puncta and ubiquitin-positive puncta normalized to soma area for neurons (G) or astrocytes (G’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 32-33 neurons and N = 23-30 astrocytes from 3 independent experiments; 7-8 DIV of coculture. Throughout the figure, neurons are circled in orange and astrocytes are circled in purple. For all graphs in all figures, small circles indicate the measurements from individual cells (e.g., the technical replicates) from each of the independent experiments; large triangles indicate the corresponding biological means from each of the independent experiments (e.g., the biological replicates); independent experiments are color-coded.
    Mouse Monoclonal Anti βiii Tubulin, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti β iii tubulin antibody mouse  (R&D Systems)
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    R&D Systems anti β iii tubulin antibody mouse
    (A) Schematic of the neuron-astrocyte coculture . (B) Representative images of the neuron-astrocyte coculture. Coculture of GFP-LC3 transgenic astrocytes and non-transgenic neurons were fixed on DIV7 of coculture (neurons were a total of DIV11) and immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), AQP4 (astrocyte marker) and <t>β3-tubulin</t> (neuron marker); nuclei were labeled with Hoechst (shown in blue in the merged image). Shown are maximum projections of z-stacks. Dashed box indicates location of the zoom-in. Scale bars, 10 µm. (C) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and LAMP1. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for GFP(LC3), p62, and LAMP1 are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate puncta co-positive for GFP(LC3) and LAMP1, or p62 and LAMP1. Empty yellow arrowheads indicate GFP(LC3) or p62 puncta that are negative for LAMP1. Scale bars, 10 µm. ( C’ ) Representative line scans of p62 and LAMP1-positive puncta in the soma of neurons and astrocytes. Asterisks denote peaks co-positive for p62 and LAMP1. (D) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and ubiquitin; nuclei (Nuc) were labeled with Hoechst. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for p62 and ubiquitin are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate colocalization between p62 puncta and ubiquitin puncta. Empty yellow arrowheads indicate p62 puncta with no ubiquitin puncta correlate. Scale bar, 10 µm. (E-E’) Quantification of the total area occupied by p62-positive puncta normalized to soma area for neurons (E) or astrocytes (E’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 37-40 neurons and N = 27-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (F-F’) Quantification of the total area occupied by ubiquitin-positive puncta normalized to soma area for neurons (F) or astrocytes (F’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 35-36 neurons and N = 29-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (G-G’) Quantification of the percentage of overlapping area between p62-positive puncta and ubiquitin-positive puncta normalized to soma area for neurons (G) or astrocytes (G’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 32-33 neurons and N = 23-30 astrocytes from 3 independent experiments; 7-8 DIV of coculture. Throughout the figure, neurons are circled in orange and astrocytes are circled in purple. For all graphs in all figures, small circles indicate the measurements from individual cells (e.g., the technical replicates) from each of the independent experiments; large triangles indicate the corresponding biological means from each of the independent experiments (e.g., the biological replicates); independent experiments are color-coded.
    Anti β Iii Tubulin Antibody Mouse, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+anti+%CE%B2+iii+tubulin/Neuron-specific+beta-III+Tubulin+Antibody/pm41827885-69-1-8
    Average 96 stars, based on 1 article reviews
    anti β iii tubulin antibody mouse - by Bioz Stars, 2026-10
    96/100 stars
    		  Buy from Supplier

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    (A) Schematic of the neuron-astrocyte coculture . (B) Representative images of the neuron-astrocyte coculture. Coculture of GFP-LC3 transgenic astrocytes and non-transgenic neurons were fixed on DIV7 of coculture (neurons were a total of DIV11) and immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), AQP4 (astrocyte marker) and β3-tubulin (neuron marker); nuclei were labeled with Hoechst (shown in blue in the merged image). Shown are maximum projections of z-stacks. Dashed box indicates location of the zoom-in. Scale bars, 10 µm. (C) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and LAMP1. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for GFP(LC3), p62, and LAMP1 are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate puncta co-positive for GFP(LC3) and LAMP1, or p62 and LAMP1. Empty yellow arrowheads indicate GFP(LC3) or p62 puncta that are negative for LAMP1. Scale bars, 10 µm. ( C’ ) Representative line scans of p62 and LAMP1-positive puncta in the soma of neurons and astrocytes. Asterisks denote peaks co-positive for p62 and LAMP1. (D) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and ubiquitin; nuclei (Nuc) were labeled with Hoechst. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for p62 and ubiquitin are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate colocalization between p62 puncta and ubiquitin puncta. Empty yellow arrowheads indicate p62 puncta with no ubiquitin puncta correlate. Scale bar, 10 µm. (E-E’) Quantification of the total area occupied by p62-positive puncta normalized to soma area for neurons (E) or astrocytes (E’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 37-40 neurons and N = 27-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (F-F’) Quantification of the total area occupied by ubiquitin-positive puncta normalized to soma area for neurons (F) or astrocytes (F’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 35-36 neurons and N = 29-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (G-G’) Quantification of the percentage of overlapping area between p62-positive puncta and ubiquitin-positive puncta normalized to soma area for neurons (G) or astrocytes (G’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 32-33 neurons and N = 23-30 astrocytes from 3 independent experiments; 7-8 DIV of coculture. Throughout the figure, neurons are circled in orange and astrocytes are circled in purple. For all graphs in all figures, small circles indicate the measurements from individual cells (e.g., the technical replicates) from each of the independent experiments; large triangles indicate the corresponding biological means from each of the independent experiments (e.g., the biological replicates); independent experiments are color-coded.

    Journal: PLOS One

    Article Title: Differential regulation of p62-ubiquitin conjugates in neurons versus astrocytes during cellular stress

    doi: 10.1371/journal.pone.0345890

    Figure Lengend Snippet: (A) Schematic of the neuron-astrocyte coculture . (B) Representative images of the neuron-astrocyte coculture. Coculture of GFP-LC3 transgenic astrocytes and non-transgenic neurons were fixed on DIV7 of coculture (neurons were a total of DIV11) and immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), AQP4 (astrocyte marker) and β3-tubulin (neuron marker); nuclei were labeled with Hoechst (shown in blue in the merged image). Shown are maximum projections of z-stacks. Dashed box indicates location of the zoom-in. Scale bars, 10 µm. (C) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and LAMP1. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for GFP(LC3), p62, and LAMP1 are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate puncta co-positive for GFP(LC3) and LAMP1, or p62 and LAMP1. Empty yellow arrowheads indicate GFP(LC3) or p62 puncta that are negative for LAMP1. Scale bars, 10 µm. ( C’ ) Representative line scans of p62 and LAMP1-positive puncta in the soma of neurons and astrocytes. Asterisks denote peaks co-positive for p62 and LAMP1. (D) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and ubiquitin; nuclei (Nuc) were labeled with Hoechst. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for p62 and ubiquitin are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate colocalization between p62 puncta and ubiquitin puncta. Empty yellow arrowheads indicate p62 puncta with no ubiquitin puncta correlate. Scale bar, 10 µm. (E-E’) Quantification of the total area occupied by p62-positive puncta normalized to soma area for neurons (E) or astrocytes (E’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 37-40 neurons and N = 27-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (F-F’) Quantification of the total area occupied by ubiquitin-positive puncta normalized to soma area for neurons (F) or astrocytes (F’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 35-36 neurons and N = 29-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (G-G’) Quantification of the percentage of overlapping area between p62-positive puncta and ubiquitin-positive puncta normalized to soma area for neurons (G) or astrocytes (G’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 32-33 neurons and N = 23-30 astrocytes from 3 independent experiments; 7-8 DIV of coculture. Throughout the figure, neurons are circled in orange and astrocytes are circled in purple. For all graphs in all figures, small circles indicate the measurements from individual cells (e.g., the technical replicates) from each of the independent experiments; large triangles indicate the corresponding biological means from each of the independent experiments (e.g., the biological replicates); independent experiments are color-coded.

    Article Snippet: Primary antibodies for immunofluorescence include mouse anti-βIII Tubulin (R&D Systems, MAB1195), rabbit anti-AQP4 (Millipore Sigma, HPA014784), chicken anti-GFP (Aves Labs, Inc., GFP-1020), rabbit anti-p62 (Abcam, Ab109012 ), mouse anti-Ubiquitin (Enzo Life Sciences, ENZ-ABS840; − and ), mouse anti-Ubiquitin (Enzo Life Sciences, BML-PW8810; , and ), and rat anti-LAMP1 (Abcam, ab25245).

    Techniques: Transgenic Assay, Marker, Labeling, Solvent, Control, Ubiquitin Proteomics