anti sca1  (Thermo Fisher)


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    Structured Review

    Thermo Fisher anti sca1
    <t>Sca1</t> ± /GFP + cells isolated from EOM preparations of the Myf5 Cre reporter line distinctively give rise to myogenic and non-myogenic progeny, respectively
    Anti Sca1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 3355 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sca1/product/Thermo Fisher
    Average 90 stars, based on 3355 article reviews
    Price from $9.99 to $1999.99
    anti sca1 - by Bioz Stars, 2020-08
    90/100 stars

    Images

    1) Product Images from "Ancestral Myf5 gene activity in periocular connective tissue identifies a subset of fibro/adipogenic progenitors but does not connote a myogenic origin"

    Article Title: Ancestral Myf5 gene activity in periocular connective tissue identifies a subset of fibro/adipogenic progenitors but does not connote a myogenic origin

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2013.08.010

    Sca1 ± /GFP + cells isolated from EOM preparations of the Myf5 Cre reporter line distinctively give rise to myogenic and non-myogenic progeny, respectively
    Figure Legend Snippet: Sca1 ± /GFP + cells isolated from EOM preparations of the Myf5 Cre reporter line distinctively give rise to myogenic and non-myogenic progeny, respectively

    Techniques Used: Isolation

    2) Product Images from "Requirement for Microglia for the Maintenance of Synaptic Function and Integrity in the Mature Retina"

    Article Title: Requirement for Microglia for the Maintenance of Synaptic Function and Integrity in the Mature Retina

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3575-15.2016

    Effect of retinal microglial depletion on retinal lamination, cell survival, and vascular structure. A , In vivo OCT assessment demonstrating horizontal linear spectral domain OCT retinal scans of control transgenic mice (TG Control) and transgenic mice following microglial depletion for 30 d (TG-depleted 30 d); insets show magnified view of retinal lamination and thickness. Overall retinal structure was preserved following sustained depletion, with clear definition of all retinal lamina at all retinal loci within the central 1.4 × 1.4 mm imaging field. INL, Inner nuclear layer; ONL, outer nuclear layer; ELM, external limiting membrane; IS/OS, junction between the inner and outer segment of the photoreceptors; RPE, retinal pigment epithelium complex. B , Mean retinal thickness in retinal areas as defined by a circular grid with concentric retinal areas centered on the optic nerve were computed. Mean retinal thickness in areas between 100 and 300 μm radial to the optic nerve (pink areas) and between 300 and 600 μm radial to the optic nerve (yellow areas) were similar between TG Control and TG Depleted, considering all four quadrants (superior, inferior, temporal, and nasal; data are represented as mean ± SEM; n = 12 eyes in 6 female TG Control animals, 16 eyes in 8 TG Depleted female animals; p = 0.18, two-way ANOVA). C , Comparison of DAPI-labeled retinal sections from adult (2- to 3-month-old) female WT C57BL6 mice, control TG mice (TG Control), and TG mice depleted of microglia for 30 d (TG Depleted 30 d) demonstrated no general atrophy of nuclear layers between groups. Retinal cell apoptosis was assessed in retinal sections using a TUNEL assay (red); TUNEL-positive cells were absent in all retinal layers in all experimental groups. Retina sections from a postnatal day (P)25 rd10 mouse retina containing apoptotic rod photoreceptors were used as a positive control. Scale bar, 50 μm. D , Labeling of retinal vasculature using isolectin-B4 (IB4) demonstrated no significant changes in TG Depleted retinas relative to WT and TG control retinas in terms of: (1) vascular patterning in retinal flat-mounts (top; insets show boxed areas at higher-magnification), and (2) laminar distribution of retinal vasculature in vibratome retinal sections (bottom). Scale bars: top, 1 mm; bottom, 50 μm.
    Figure Legend Snippet: Effect of retinal microglial depletion on retinal lamination, cell survival, and vascular structure. A , In vivo OCT assessment demonstrating horizontal linear spectral domain OCT retinal scans of control transgenic mice (TG Control) and transgenic mice following microglial depletion for 30 d (TG-depleted 30 d); insets show magnified view of retinal lamination and thickness. Overall retinal structure was preserved following sustained depletion, with clear definition of all retinal lamina at all retinal loci within the central 1.4 × 1.4 mm imaging field. INL, Inner nuclear layer; ONL, outer nuclear layer; ELM, external limiting membrane; IS/OS, junction between the inner and outer segment of the photoreceptors; RPE, retinal pigment epithelium complex. B , Mean retinal thickness in retinal areas as defined by a circular grid with concentric retinal areas centered on the optic nerve were computed. Mean retinal thickness in areas between 100 and 300 μm radial to the optic nerve (pink areas) and between 300 and 600 μm radial to the optic nerve (yellow areas) were similar between TG Control and TG Depleted, considering all four quadrants (superior, inferior, temporal, and nasal; data are represented as mean ± SEM; n = 12 eyes in 6 female TG Control animals, 16 eyes in 8 TG Depleted female animals; p = 0.18, two-way ANOVA). C , Comparison of DAPI-labeled retinal sections from adult (2- to 3-month-old) female WT C57BL6 mice, control TG mice (TG Control), and TG mice depleted of microglia for 30 d (TG Depleted 30 d) demonstrated no general atrophy of nuclear layers between groups. Retinal cell apoptosis was assessed in retinal sections using a TUNEL assay (red); TUNEL-positive cells were absent in all retinal layers in all experimental groups. Retina sections from a postnatal day (P)25 rd10 mouse retina containing apoptotic rod photoreceptors were used as a positive control. Scale bar, 50 μm. D , Labeling of retinal vasculature using isolectin-B4 (IB4) demonstrated no significant changes in TG Depleted retinas relative to WT and TG control retinas in terms of: (1) vascular patterning in retinal flat-mounts (top; insets show boxed areas at higher-magnification), and (2) laminar distribution of retinal vasculature in vibratome retinal sections (bottom). Scale bars: top, 1 mm; bottom, 50 μm.

    Techniques Used: In Vivo, Transgenic Assay, Mouse Assay, Imaging, Labeling, TUNEL Assay, Positive Control

    3) Product Images from "Muc2 Protects against Lethal Infectious Colitis by Disassociating Pathogenic and Commensal Bacteria from the Colonic Mucosa"

    Article Title: Muc2 Protects against Lethal Infectious Colitis by Disassociating Pathogenic and Commensal Bacteria from the Colonic Mucosa

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000902

    C. rodentium infection results in increased mucin secretion during infection. A. Representative PAS/Haematoxylin staining of Carnoy's fixed rectal sections from uninfected (left panel) and C. rodentium -infected mice (right panel). Arrow points to luminal mucus. Original magnification = 100×. Scale bar = 100 µm. B. Total counts per minute (CPM) of [ 3 H]-glucosamine labeled glycoproteins found in colorectal secretions 3.5 hrs post-injection from uninfected and infected (6 DPI) WT mice. Results are representative of 2 independent infections containing 5 mice per group. C. Plot of liquid scintillation counts of fractions containing [ 3 H] activity after total secretions were subjected to gel filtration on a Sepharose 4B chromatography column. This graph is representative of 2 independent infections with 5 mice per group. D. Graph of total CPMs of void volumes of S4B-fractionated mucins as described in D. Data represents the mean of the average of 2 independent experiments, each with 5 mice per group. Error bars = SEM. E. Combined epifluorescent staining for mucus using the lectin UEA-1 (red), and C. rodentium LPS (green), and cellular DNA (blue) using DAPI as a counterstain in heavily infected (6 DPI) regions of the colorectal mucosa from WT and Muc2 −/− mice, as indicated. Individual C. rodentium (arrowhead, inset “a”) can be seen in mucus overlying a single layer of C. rodentium on the mucosal surface of a WT mouse. A C. rodentium microcolony (white arrow) can be seen in vicinity of a Muc2/mucus-deficient environment as indicated by the absence of mucus in the crypt lumens in Muc2 −/− mice compared to WT mice (yellow arrow). Original magnification = 200×. Scale bar = 50 µm.
    Figure Legend Snippet: C. rodentium infection results in increased mucin secretion during infection. A. Representative PAS/Haematoxylin staining of Carnoy's fixed rectal sections from uninfected (left panel) and C. rodentium -infected mice (right panel). Arrow points to luminal mucus. Original magnification = 100×. Scale bar = 100 µm. B. Total counts per minute (CPM) of [ 3 H]-glucosamine labeled glycoproteins found in colorectal secretions 3.5 hrs post-injection from uninfected and infected (6 DPI) WT mice. Results are representative of 2 independent infections containing 5 mice per group. C. Plot of liquid scintillation counts of fractions containing [ 3 H] activity after total secretions were subjected to gel filtration on a Sepharose 4B chromatography column. This graph is representative of 2 independent infections with 5 mice per group. D. Graph of total CPMs of void volumes of S4B-fractionated mucins as described in D. Data represents the mean of the average of 2 independent experiments, each with 5 mice per group. Error bars = SEM. E. Combined epifluorescent staining for mucus using the lectin UEA-1 (red), and C. rodentium LPS (green), and cellular DNA (blue) using DAPI as a counterstain in heavily infected (6 DPI) regions of the colorectal mucosa from WT and Muc2 −/− mice, as indicated. Individual C. rodentium (arrowhead, inset “a”) can be seen in mucus overlying a single layer of C. rodentium on the mucosal surface of a WT mouse. A C. rodentium microcolony (white arrow) can be seen in vicinity of a Muc2/mucus-deficient environment as indicated by the absence of mucus in the crypt lumens in Muc2 −/− mice compared to WT mice (yellow arrow). Original magnification = 200×. Scale bar = 50 µm.

    Techniques Used: Infection, Staining, Mouse Assay, Labeling, Injection, Activity Assay, Filtration, Chromatography

    4) Product Images from "A role for SOX9 in post-transcriptional processes: insights from the amphibian oocyte"

    Article Title: A role for SOX9 in post-transcriptional processes: insights from the amphibian oocyte

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25356-1

    SOX9 protein does not bind the chromosome axis of the LBCs. Immunostaining of P. waltl nuclear spreads using the anti-Cter-SOX9 antibody (green, Alexa 488 IgG) and counterstained with Hoechst (red). ( A ) Fluorescent micrograph (a) and its corresponding negative (b) showing a strong SOX9 immunostaining detected in the close vicinity of the chromosome axis over the thickest region of the lateral loops ( B ) Nuclear spreads from an oocyte at the end of stage VI. The SOX9 protein was not detected at the level of the chromosome axis where it was devoid of lateral loops (white arrows, merge panel). Scale bar for all micrographs: 10 μm.
    Figure Legend Snippet: SOX9 protein does not bind the chromosome axis of the LBCs. Immunostaining of P. waltl nuclear spreads using the anti-Cter-SOX9 antibody (green, Alexa 488 IgG) and counterstained with Hoechst (red). ( A ) Fluorescent micrograph (a) and its corresponding negative (b) showing a strong SOX9 immunostaining detected in the close vicinity of the chromosome axis over the thickest region of the lateral loops ( B ) Nuclear spreads from an oocyte at the end of stage VI. The SOX9 protein was not detected at the level of the chromosome axis where it was devoid of lateral loops (white arrows, merge panel). Scale bar for all micrographs: 10 μm.

    Techniques Used: Immunostaining

    Subnuclear localization of the SOX9 protein in the GV of X. tropicalis , X. laevis and P. waltl . Fluorescent and corresponding phase contrast micrographs of nuclear spreads showing one LBC of X. tropicalis and X. laevis from stage V-VI oocyte, and part of a P. waltl LBC from stages II and V-VI oocyte. Nuclear spreads were immunostained with the anti-Cter-SOX9 antibody (green, Alexa 488 IgG) and counterstained with Hoechst (red). The Hoechst dye stained the chromosome DNA axis and not that of the decondensed lateral loops. The antibody labeled the LBCs, and the nuclear bodies either attached to them (empty arrowheads) or free in the nucleoplasm (full arrowheads). Note that the immunostaining of lateral loops was very clear in P. waltl LBCs from stage II oocyte, because of their extension and density. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls with the secondary antibody (tagged with Alexa fluor 488) alone. Wide field Leica microscope. Scale bar for all micrographs: 10 μm.
    Figure Legend Snippet: Subnuclear localization of the SOX9 protein in the GV of X. tropicalis , X. laevis and P. waltl . Fluorescent and corresponding phase contrast micrographs of nuclear spreads showing one LBC of X. tropicalis and X. laevis from stage V-VI oocyte, and part of a P. waltl LBC from stages II and V-VI oocyte. Nuclear spreads were immunostained with the anti-Cter-SOX9 antibody (green, Alexa 488 IgG) and counterstained with Hoechst (red). The Hoechst dye stained the chromosome DNA axis and not that of the decondensed lateral loops. The antibody labeled the LBCs, and the nuclear bodies either attached to them (empty arrowheads) or free in the nucleoplasm (full arrowheads). Note that the immunostaining of lateral loops was very clear in P. waltl LBCs from stage II oocyte, because of their extension and density. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls with the secondary antibody (tagged with Alexa fluor 488) alone. Wide field Leica microscope. Scale bar for all micrographs: 10 μm.

    Techniques Used: Staining, Labeling, Immunostaining, Fluorescence, Microscopy

    Super-resolution images of a P. waltl lateral loop immunostained for CELF1 [mAb3B1 (red, Alexa 568 IgG)] and SOX9 [anti-Cter-SOX9 antibody (green, Alexa 488 IgG)]. The RNP matrix exhibited a pattern of granules immunostained for SOX9 and CELF1. The merged panel showed that the SOX9 and CELF1 granules did not colocalize. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls with the secondary antibodies alone. Structured Illumination Microscopy (SIM). The image in the boxed area corresponded to the phase contrast image obtained from Wide field Leica microscopy. Scale bar: 10 μm.
    Figure Legend Snippet: Super-resolution images of a P. waltl lateral loop immunostained for CELF1 [mAb3B1 (red, Alexa 568 IgG)] and SOX9 [anti-Cter-SOX9 antibody (green, Alexa 488 IgG)]. The RNP matrix exhibited a pattern of granules immunostained for SOX9 and CELF1. The merged panel showed that the SOX9 and CELF1 granules did not colocalize. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls with the secondary antibodies alone. Structured Illumination Microscopy (SIM). The image in the boxed area corresponded to the phase contrast image obtained from Wide field Leica microscopy. Scale bar: 10 μm.

    Techniques Used: Fluorescence, Microscopy

    Localization of SOX9 to the matrix of lateral loops is RNA-dependent. Nuclear spreads of X. laevis were immunostained for SOX9 [anti-Cter-SOX9 antibody (green, Alexa 488 IgG)] and SR proteins [mAb1H4 (red, Alexa 568 IgG)]. ( a – e ’) LBC from untreated nuclear spreads. ( f – j ’) LBC from nuclear spread digested with RNase A before immunostaining. ( a ’– e ’) Enlarged images of the regions marked by white boxes ( a–e ) show several lateral loops immunostained with SOX9 and SR antibodies. The arrowheads point to terminal granules. ( f ’– j ’) enlarged images of the regions marked by white boxes ( f–j ) show one lateral loop not stained with SOX9 and SR antibodies after digestion with Rnase A. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls (the secondary antibodies only). Wide field Leica microscopy. Scale bar: 10 μm.
    Figure Legend Snippet: Localization of SOX9 to the matrix of lateral loops is RNA-dependent. Nuclear spreads of X. laevis were immunostained for SOX9 [anti-Cter-SOX9 antibody (green, Alexa 488 IgG)] and SR proteins [mAb1H4 (red, Alexa 568 IgG)]. ( a – e ’) LBC from untreated nuclear spreads. ( f – j ’) LBC from nuclear spread digested with RNase A before immunostaining. ( a ’– e ’) Enlarged images of the regions marked by white boxes ( a–e ) show several lateral loops immunostained with SOX9 and SR antibodies. The arrowheads point to terminal granules. ( f ’– j ’) enlarged images of the regions marked by white boxes ( f–j ) show one lateral loop not stained with SOX9 and SR antibodies after digestion with Rnase A. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls (the secondary antibodies only). Wide field Leica microscopy. Scale bar: 10 μm.

    Techniques Used: Immunostaining, Staining, Fluorescence, Microscopy

    Immunostaining of SOX9 after inhibition of transcription. Oocytes of P. waltl were treated or not with α-Amanitin or Actinomycin D (Act D). Phase contrast and corresponding fluorescent micrographs of nuclear spreads that were stained with the anti-Cter SOX9 antibody (green, Alexa 488 IgG) and counterstained with Hoechst (red) to show the chromosome axis. In contrast to the LBCs from the not-treated oocytes, those from the oocytes incubated with α-amanitin or Act D were devoid of their lateral loops and and the chromosome axis lacked SOX9 staining. Note that the spheres (arrowheads) known to be storage sites for transcription and post-transcription factors were strongly stained. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls with the secondary antibodies alone. Wide field Leica microscope. Scale bar for all micrographs: 15 μm.
    Figure Legend Snippet: Immunostaining of SOX9 after inhibition of transcription. Oocytes of P. waltl were treated or not with α-Amanitin or Actinomycin D (Act D). Phase contrast and corresponding fluorescent micrographs of nuclear spreads that were stained with the anti-Cter SOX9 antibody (green, Alexa 488 IgG) and counterstained with Hoechst (red) to show the chromosome axis. In contrast to the LBCs from the not-treated oocytes, those from the oocytes incubated with α-amanitin or Act D were devoid of their lateral loops and and the chromosome axis lacked SOX9 staining. Note that the spheres (arrowheads) known to be storage sites for transcription and post-transcription factors were strongly stained. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls with the secondary antibodies alone. Wide field Leica microscope. Scale bar for all micrographs: 15 μm.

    Techniques Used: Immunostaining, Inhibition, Activated Clotting Time Assay, Staining, Incubation, Fluorescence, Microscopy

    5) Product Images from "Immune Stimulation Using a Gut Microbe-Based Immunotherapy Reduces Disease Pathology and Improves Barrier Function in Ulcerative Colitis"

    Article Title: Immune Stimulation Using a Gut Microbe-Based Immunotherapy Reduces Disease Pathology and Improves Barrier Function in Ulcerative Colitis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02211

    QBECO treatment reduced disease severity in Muc-2 −/− mice. Muc2 −/− mice were injected subcutaneously with placebo or QBECO every other day for 30 days and colonic tissue was collected for histology grading (A,B) . Colonic tissues were also stained with (C) anti-Ly6G to assess neutrophil number and (D) anti-CD3 to assess the number of T cells in colonic tissues. Control n = 15 QBECO: n = 10, mean ± SEM, * p
    Figure Legend Snippet: QBECO treatment reduced disease severity in Muc-2 −/− mice. Muc2 −/− mice were injected subcutaneously with placebo or QBECO every other day for 30 days and colonic tissue was collected for histology grading (A,B) . Colonic tissues were also stained with (C) anti-Ly6G to assess neutrophil number and (D) anti-CD3 to assess the number of T cells in colonic tissues. Control n = 15 QBECO: n = 10, mean ± SEM, * p

    Techniques Used: Mouse Assay, Injection, Staining

    6) Product Images from "Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks"

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    Journal: bioRxiv

    doi: 10.1101/864769

    Expression of EGFP-TWF-1 rescues the twf1/twf2 knockout phenotype. Representative images of twf1/twf2-KO cells expressing EGFP-TWF-1 and stained with AlexaFluor-647 phalloidin and DAPI to visualize F-actin and nuclei, respectively. Scale bars = 20 μ m.
    Figure Legend Snippet: Expression of EGFP-TWF-1 rescues the twf1/twf2 knockout phenotype. Representative images of twf1/twf2-KO cells expressing EGFP-TWF-1 and stained with AlexaFluor-647 phalloidin and DAPI to visualize F-actin and nuclei, respectively. Scale bars = 20 μ m.

    Techniques Used: Expressing, Knock-Out, Staining

    Twinfilin-1/twinfilin-2 knockout leads to an accumulation of F-actin on endosomes at the perinuclear region. (A) Phalloidin staining of wild-type B16-F1, and (B) twf1/twf2-KO cells after 7.5 min uptake of 20 μ g/ml AlexaFluor-647 transferrin. Scale bar = 20 μ m. The dotted square indicates the perinuclear region magnified in the insert. (C) Mean F-actin intensities in transferrin-positive endosomes of wild-type and twf1/twf2-KO cells as measured from AlexaFluor-555 phalloidin stained cells after 7.5 min intake of 20 μ g/ml AlexaFluor-647 phalloidin. Numbers of measured cells were: B16-F1 wt = 2,518, twf1/2-KO-g3 = 3,637, twf1/2-KO-g3 + EGFP-TWF1 = 158. Statistical significances were calculated with Mann-Whitney two-tailed test. ****, p
    Figure Legend Snippet: Twinfilin-1/twinfilin-2 knockout leads to an accumulation of F-actin on endosomes at the perinuclear region. (A) Phalloidin staining of wild-type B16-F1, and (B) twf1/twf2-KO cells after 7.5 min uptake of 20 μ g/ml AlexaFluor-647 transferrin. Scale bar = 20 μ m. The dotted square indicates the perinuclear region magnified in the insert. (C) Mean F-actin intensities in transferrin-positive endosomes of wild-type and twf1/twf2-KO cells as measured from AlexaFluor-555 phalloidin stained cells after 7.5 min intake of 20 μ g/ml AlexaFluor-647 phalloidin. Numbers of measured cells were: B16-F1 wt = 2,518, twf1/2-KO-g3 = 3,637, twf1/2-KO-g3 + EGFP-TWF1 = 158. Statistical significances were calculated with Mann-Whitney two-tailed test. ****, p

    Techniques Used: Knock-Out, Staining, MANN-WHITNEY, Two Tailed Test

    7) Product Images from "Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks"

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    Journal: bioRxiv

    doi: 10.1101/864769

    Examples of high-content and lamellipodia protrusion analysis. (A) Representative images of segmentation procedure in high-content image analysis. Wild-type and twinfilin-deficient B16-F1 cells were stained with DAPI and CellMask Deep Red to segment nuclei (outlined with blue) and cytoplasm (outlined with yellow). The Arp2/3-complex positive structures were detected with anti-p34 antibody staining and used as a mask for the Arp2/3-complex positive F-actin structures (the most-right panel). F-actin was stained with AlexaFluor-568 phalloidin. Cells touching the border of images were excluded from analysis. (B) A representative example of twf1/twf2-KO cell migrating on laminin coated glass imaged with DIC. Direction of migration is indicated with an arrow and kymographs were generated with line drawn across the lamellipodium as indicated with dotted red line. (C) Representative examples of kymographs generated from DIC time-lapse images of wild-type, twf1/twf2 knockout, and EGFP-TWF-1 rescue cells. Protrusions velocities were measured from the overall cell front protrusion as indicated with dotted red lines.
    Figure Legend Snippet: Examples of high-content and lamellipodia protrusion analysis. (A) Representative images of segmentation procedure in high-content image analysis. Wild-type and twinfilin-deficient B16-F1 cells were stained with DAPI and CellMask Deep Red to segment nuclei (outlined with blue) and cytoplasm (outlined with yellow). The Arp2/3-complex positive structures were detected with anti-p34 antibody staining and used as a mask for the Arp2/3-complex positive F-actin structures (the most-right panel). F-actin was stained with AlexaFluor-568 phalloidin. Cells touching the border of images were excluded from analysis. (B) A representative example of twf1/twf2-KO cell migrating on laminin coated glass imaged with DIC. Direction of migration is indicated with an arrow and kymographs were generated with line drawn across the lamellipodium as indicated with dotted red line. (C) Representative examples of kymographs generated from DIC time-lapse images of wild-type, twf1/twf2 knockout, and EGFP-TWF-1 rescue cells. Protrusions velocities were measured from the overall cell front protrusion as indicated with dotted red lines.

    Techniques Used: Staining, Migration, Generated, Knock-Out

    8) Product Images from "A role for SOX9 in post-transcriptional processes: insights from the amphibian oocyte"

    Article Title: A role for SOX9 in post-transcriptional processes: insights from the amphibian oocyte

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25356-1

    SOX9 protein does not bind the chromosome axis of the LBCs. Immunostaining of P. waltl nuclear spreads using the anti-Cter-SOX9 antibody (green, Alexa 488 IgG) and counterstained with Hoechst (red). ( A ) Fluorescent micrograph (a) and its corresponding negative (b) showing a strong SOX9 immunostaining detected in the close vicinity of the chromosome axis over the thickest region of the lateral loops ( B ) Nuclear spreads from an oocyte at the end of stage VI. The SOX9 protein was not detected at the level of the chromosome axis where it was devoid of lateral loops (white arrows, merge panel). Scale bar for all micrographs: 10 μm.
    Figure Legend Snippet: SOX9 protein does not bind the chromosome axis of the LBCs. Immunostaining of P. waltl nuclear spreads using the anti-Cter-SOX9 antibody (green, Alexa 488 IgG) and counterstained with Hoechst (red). ( A ) Fluorescent micrograph (a) and its corresponding negative (b) showing a strong SOX9 immunostaining detected in the close vicinity of the chromosome axis over the thickest region of the lateral loops ( B ) Nuclear spreads from an oocyte at the end of stage VI. The SOX9 protein was not detected at the level of the chromosome axis where it was devoid of lateral loops (white arrows, merge panel). Scale bar for all micrographs: 10 μm.

    Techniques Used: Immunostaining

    Subnuclear localization of the SOX9 protein in the GV of X. tropicalis , X. laevis and P. waltl . Fluorescent and corresponding phase contrast micrographs of nuclear spreads showing one LBC of X. tropicalis and X. laevis from stage V-VI oocyte, and part of a P. waltl LBC from stages II and V-VI oocyte. Nuclear spreads were immunostained with the anti-Cter-SOX9 antibody (green, Alexa 488 IgG) and counterstained with Hoechst (red). The Hoechst dye stained the chromosome DNA axis and not that of the decondensed lateral loops. The antibody labeled the LBCs, and the nuclear bodies either attached to them (empty arrowheads) or free in the nucleoplasm (full arrowheads). Note that the immunostaining of lateral loops was very clear in P. waltl LBCs from stage II oocyte, because of their extension and density. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls with the secondary antibody (tagged with Alexa fluor 488) alone. Wide field Leica microscope. Scale bar for all micrographs: 10 μm.
    Figure Legend Snippet: Subnuclear localization of the SOX9 protein in the GV of X. tropicalis , X. laevis and P. waltl . Fluorescent and corresponding phase contrast micrographs of nuclear spreads showing one LBC of X. tropicalis and X. laevis from stage V-VI oocyte, and part of a P. waltl LBC from stages II and V-VI oocyte. Nuclear spreads were immunostained with the anti-Cter-SOX9 antibody (green, Alexa 488 IgG) and counterstained with Hoechst (red). The Hoechst dye stained the chromosome DNA axis and not that of the decondensed lateral loops. The antibody labeled the LBCs, and the nuclear bodies either attached to them (empty arrowheads) or free in the nucleoplasm (full arrowheads). Note that the immunostaining of lateral loops was very clear in P. waltl LBCs from stage II oocyte, because of their extension and density. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls with the secondary antibody (tagged with Alexa fluor 488) alone. Wide field Leica microscope. Scale bar for all micrographs: 10 μm.

    Techniques Used: Staining, Labeling, Immunostaining, Fluorescence, Microscopy

    Super-resolution images of a P. waltl lateral loop immunostained for CELF1 [mAb3B1 (red, Alexa 568 IgG)] and SOX9 [anti-Cter-SOX9 antibody (green, Alexa 488 IgG)]. The RNP matrix exhibited a pattern of granules immunostained for SOX9 and CELF1. The merged panel showed that the SOX9 and CELF1 granules did not colocalize. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls with the secondary antibodies alone. Structured Illumination Microscopy (SIM). The image in the boxed area corresponded to the phase contrast image obtained from Wide field Leica microscopy. Scale bar: 10 μm.
    Figure Legend Snippet: Super-resolution images of a P. waltl lateral loop immunostained for CELF1 [mAb3B1 (red, Alexa 568 IgG)] and SOX9 [anti-Cter-SOX9 antibody (green, Alexa 488 IgG)]. The RNP matrix exhibited a pattern of granules immunostained for SOX9 and CELF1. The merged panel showed that the SOX9 and CELF1 granules did not colocalize. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls with the secondary antibodies alone. Structured Illumination Microscopy (SIM). The image in the boxed area corresponded to the phase contrast image obtained from Wide field Leica microscopy. Scale bar: 10 μm.

    Techniques Used: Fluorescence, Microscopy

    Localization of SOX9 to the matrix of lateral loops is RNA-dependent. Nuclear spreads of X. laevis were immunostained for SOX9 [anti-Cter-SOX9 antibody (green, Alexa 488 IgG)] and SR proteins [mAb1H4 (red, Alexa 568 IgG)]. ( a – e ’) LBC from untreated nuclear spreads. ( f – j ’) LBC from nuclear spread digested with RNase A before immunostaining. ( a ’– e ’) Enlarged images of the regions marked by white boxes ( a–e ) show several lateral loops immunostained with SOX9 and SR antibodies. The arrowheads point to terminal granules. ( f ’– j ’) enlarged images of the regions marked by white boxes ( f–j ) show one lateral loop not stained with SOX9 and SR antibodies after digestion with Rnase A. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls (the secondary antibodies only). Wide field Leica microscopy. Scale bar: 10 μm.
    Figure Legend Snippet: Localization of SOX9 to the matrix of lateral loops is RNA-dependent. Nuclear spreads of X. laevis were immunostained for SOX9 [anti-Cter-SOX9 antibody (green, Alexa 488 IgG)] and SR proteins [mAb1H4 (red, Alexa 568 IgG)]. ( a – e ’) LBC from untreated nuclear spreads. ( f – j ’) LBC from nuclear spread digested with RNase A before immunostaining. ( a ’– e ’) Enlarged images of the regions marked by white boxes ( a–e ) show several lateral loops immunostained with SOX9 and SR antibodies. The arrowheads point to terminal granules. ( f ’– j ’) enlarged images of the regions marked by white boxes ( f–j ) show one lateral loop not stained with SOX9 and SR antibodies after digestion with Rnase A. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls (the secondary antibodies only). Wide field Leica microscopy. Scale bar: 10 μm.

    Techniques Used: Immunostaining, Staining, Fluorescence, Microscopy

    Immunostaining of SOX9 after inhibition of transcription. Oocytes of P. waltl were treated or not with α-Amanitin or Actinomycin D (Act D). Phase contrast and corresponding fluorescent micrographs of nuclear spreads that were stained with the anti-Cter SOX9 antibody (green, Alexa 488 IgG) and counterstained with Hoechst (red) to show the chromosome axis. In contrast to the LBCs from the not-treated oocytes, those from the oocytes incubated with α-amanitin or Act D were devoid of their lateral loops and and the chromosome axis lacked SOX9 staining. Note that the spheres (arrowheads) known to be storage sites for transcription and post-transcription factors were strongly stained. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls with the secondary antibodies alone. Wide field Leica microscope. Scale bar for all micrographs: 15 μm.
    Figure Legend Snippet: Immunostaining of SOX9 after inhibition of transcription. Oocytes of P. waltl were treated or not with α-Amanitin or Actinomycin D (Act D). Phase contrast and corresponding fluorescent micrographs of nuclear spreads that were stained with the anti-Cter SOX9 antibody (green, Alexa 488 IgG) and counterstained with Hoechst (red) to show the chromosome axis. In contrast to the LBCs from the not-treated oocytes, those from the oocytes incubated with α-amanitin or Act D were devoid of their lateral loops and and the chromosome axis lacked SOX9 staining. Note that the spheres (arrowheads) known to be storage sites for transcription and post-transcription factors were strongly stained. The intensity of fluorescence in the different experiments was normalized with respect to their relevant controls with the secondary antibodies alone. Wide field Leica microscope. Scale bar for all micrographs: 15 μm.

    Techniques Used: Immunostaining, Inhibition, Activated Clotting Time Assay, Staining, Incubation, Fluorescence, Microscopy

    Immunostaining of Pol II and SOX9 in P. waltl LBCs. Nuclear spreads were immunostained for SOX9 with the anti-Cter-SOX9 antibody (red, Alexa 568 IgG) and for Pol II with the mAbH14 (green, Alexa 488 IgM). Pol II immunostaining was continuous over the loop axis while that of SOX9 was concentrated on granules distributed above the loop axis. This double immunostaining pattern was particularly visible on the two lateral loops indicated by arrows in the boxed areas. The intensity of fluorescence in this experiment was normalized with respect to its relevant controls (i.e., secondary antibodies alone). The image was deconvoluted (see Figure S7 for the non deconvoluted image). Wide field Leica microscope. Scale bar: 10 μm.
    Figure Legend Snippet: Immunostaining of Pol II and SOX9 in P. waltl LBCs. Nuclear spreads were immunostained for SOX9 with the anti-Cter-SOX9 antibody (red, Alexa 568 IgG) and for Pol II with the mAbH14 (green, Alexa 488 IgM). Pol II immunostaining was continuous over the loop axis while that of SOX9 was concentrated on granules distributed above the loop axis. This double immunostaining pattern was particularly visible on the two lateral loops indicated by arrows in the boxed areas. The intensity of fluorescence in this experiment was normalized with respect to its relevant controls (i.e., secondary antibodies alone). The image was deconvoluted (see Figure S7 for the non deconvoluted image). Wide field Leica microscope. Scale bar: 10 μm.

    Techniques Used: Immunostaining, Double Immunostaining, Fluorescence, Microscopy

    9) Product Images from "Absence of TGFβ signaling in retinal microglia induces retinal degeneration and exacerbates choroidal neovascularization"

    Article Title: Absence of TGFβ signaling in retinal microglia induces retinal degeneration and exacerbates choroidal neovascularization

    Journal: eLife

    doi: 10.7554/eLife.42049

    Specific TGFBR2 ablation in retinal microglia induces alterations in microglial morphology in the IPL and SRS. ( A, B ) Comparison of retinal flat-mounts from untreated wild type animals, control and TG animals administered tamoxifen (TMX) at the 3 week post-TMX time point demonstrated a transition from a ramified to a branched morphology in TG animals, but in control animals. Images of microglia at the level of the inner plexiform layer (IPL) ( A ) and subretinal space (SRS) ( B ) are shown. Microglia in TG animals in the IPL transitioned to a branched morphology, adhering closely to retinal vessels but those in WT and TMX-treated control animals remained unchanged. Insets ( yellow boxes ) show examples at higher magnification. ( C ) Cx3cr1 CreER/+ , TGFBR2 flox/flox , Ai14/+ mice, in which tamoxifen-induced Cre recombination induced specific ablation of TGFBR2 and expression of tdTomato in CX3CR1-expressing microglia and monocytes, were analyzed 4 months after tamoxifen to allow sufficient time for systemic monocytes to be turned over and become tdTomato-negative. All IBA1+ microglia in both the IPL and OPL demonstrated tdTomato labeling, indicating that ongoing monocyte infiltration did not contribute to the population of TGFBR2-deficient myeloid cells in the retina. ( D ) Comparison of retinal flat-mounts from control and TG animals administered tamoxifen (TMX) at the level of the OPL revealed that P2RY12 immunopositive endogenous microglia in TG animals all demonstrated a progressive loss of ramification and an acquisition of Ki67-immunopositivity in the first 3 days following TMX administration, indicating that morphological transformation and cellular proliferation occurred in resident microglia. Infiltrating monocytes, (which would have been CD11b+, P2RY12-) were not detected at any timepoint. Deramified microglia gradually decreased in P2RY12 immunopositivity at 4 days and was undetectable by 7 days following TMX. Scale bars = 100 µm.
    Figure Legend Snippet: Specific TGFBR2 ablation in retinal microglia induces alterations in microglial morphology in the IPL and SRS. ( A, B ) Comparison of retinal flat-mounts from untreated wild type animals, control and TG animals administered tamoxifen (TMX) at the 3 week post-TMX time point demonstrated a transition from a ramified to a branched morphology in TG animals, but in control animals. Images of microglia at the level of the inner plexiform layer (IPL) ( A ) and subretinal space (SRS) ( B ) are shown. Microglia in TG animals in the IPL transitioned to a branched morphology, adhering closely to retinal vessels but those in WT and TMX-treated control animals remained unchanged. Insets ( yellow boxes ) show examples at higher magnification. ( C ) Cx3cr1 CreER/+ , TGFBR2 flox/flox , Ai14/+ mice, in which tamoxifen-induced Cre recombination induced specific ablation of TGFBR2 and expression of tdTomato in CX3CR1-expressing microglia and monocytes, were analyzed 4 months after tamoxifen to allow sufficient time for systemic monocytes to be turned over and become tdTomato-negative. All IBA1+ microglia in both the IPL and OPL demonstrated tdTomato labeling, indicating that ongoing monocyte infiltration did not contribute to the population of TGFBR2-deficient myeloid cells in the retina. ( D ) Comparison of retinal flat-mounts from control and TG animals administered tamoxifen (TMX) at the level of the OPL revealed that P2RY12 immunopositive endogenous microglia in TG animals all demonstrated a progressive loss of ramification and an acquisition of Ki67-immunopositivity in the first 3 days following TMX administration, indicating that morphological transformation and cellular proliferation occurred in resident microglia. Infiltrating monocytes, (which would have been CD11b+, P2RY12-) were not detected at any timepoint. Deramified microglia gradually decreased in P2RY12 immunopositivity at 4 days and was undetectable by 7 days following TMX. Scale bars = 100 µm.

    Techniques Used: Mouse Assay, Expressing, Labeling, Transformation Assay

    10) Product Images from "Absence of TGFβ signaling in retinal microglia induces retinal degeneration and exacerbates choroidal neovascularization"

    Article Title: Absence of TGFβ signaling in retinal microglia induces retinal degeneration and exacerbates choroidal neovascularization

    Journal: eLife

    doi: 10.7554/eLife.42049

    TGFBR2 ablation in retinal microglia induces Müller cell gliosis in the retina. ( A ) Immunohistochemical analysis demonstrates upregulation of immunopositivity to GFAP 3 weeks post-TMX in TG animals relative to control animals. GFAP immunopositivity was localized to glutamine synthetase (GS)-labeled Müller cell processes, indicating the induction of Müller cell gliosis. Scale bar = 50 µm. ( B ) qPCR analysis of retinas isolated from control and TG animals 2 and 8 weeks post-TMX demonstrates a significant upregulation of GFAP mRNA expression following TGFBR2 ablation in retinal microglia. Graphical data are presented as means ± SEM; p values are from one-way analysis of variance (ANOVA) and Sidak’s multiple comparison test, n = 3 animals of mixed sex in each group.( C ) RT-PCR analysis of retinal expression of genes associated with A1- and A2-specific astrocytic gliosis following microglial TGFBR2 ablation found progressive upregulation of A1-associated transcripts relative to control, while A2-associated transcripts were relatively unchanged (numbers indicate means, *, **, *** indicate p values
    Figure Legend Snippet: TGFBR2 ablation in retinal microglia induces Müller cell gliosis in the retina. ( A ) Immunohistochemical analysis demonstrates upregulation of immunopositivity to GFAP 3 weeks post-TMX in TG animals relative to control animals. GFAP immunopositivity was localized to glutamine synthetase (GS)-labeled Müller cell processes, indicating the induction of Müller cell gliosis. Scale bar = 50 µm. ( B ) qPCR analysis of retinas isolated from control and TG animals 2 and 8 weeks post-TMX demonstrates a significant upregulation of GFAP mRNA expression following TGFBR2 ablation in retinal microglia. Graphical data are presented as means ± SEM; p values are from one-way analysis of variance (ANOVA) and Sidak’s multiple comparison test, n = 3 animals of mixed sex in each group.( C ) RT-PCR analysis of retinal expression of genes associated with A1- and A2-specific astrocytic gliosis following microglial TGFBR2 ablation found progressive upregulation of A1-associated transcripts relative to control, while A2-associated transcripts were relatively unchanged (numbers indicate means, *, **, *** indicate p values

    Techniques Used: Immunohistochemistry, Labeling, Real-time Polymerase Chain Reaction, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction

    Specific TGFBR2 ablation in retinal microglia induces rapid and progressive changes in microglial morphology and distribution. The time course of morphological changes in retinal microglia following tamoxifen (TMX)-induced ablation of TGFBR2 expression was followed using immunohistochemical analysis in retinal flat-mounts. Panels show changes at the level of the OPL; microglia were labeled using an antibody to IBA1 and retinal vessels labeled with IB4. Gliotic changes in radial Müller glia processes were marked using an antibody to GFAP. At 1 day following TMX administration, a slight reduction in ramification in microglia processes was observed. From 2–5 days post-TMX, a further decrease in microglial ramification and an increase in microglia numbers were detected. From 3–10 weeks post-TMX, retinal microglia transitioned to a branched morphology, demonstrating a close fasciculation with the retinal vasculature. GFAP immunopositivity in Müller glia was prominently upregulated at this time. Scale bar = 100 µm.
    Figure Legend Snippet: Specific TGFBR2 ablation in retinal microglia induces rapid and progressive changes in microglial morphology and distribution. The time course of morphological changes in retinal microglia following tamoxifen (TMX)-induced ablation of TGFBR2 expression was followed using immunohistochemical analysis in retinal flat-mounts. Panels show changes at the level of the OPL; microglia were labeled using an antibody to IBA1 and retinal vessels labeled with IB4. Gliotic changes in radial Müller glia processes were marked using an antibody to GFAP. At 1 day following TMX administration, a slight reduction in ramification in microglia processes was observed. From 2–5 days post-TMX, a further decrease in microglial ramification and an increase in microglia numbers were detected. From 3–10 weeks post-TMX, retinal microglia transitioned to a branched morphology, demonstrating a close fasciculation with the retinal vasculature. GFAP immunopositivity in Müller glia was prominently upregulated at this time. Scale bar = 100 µm.

    Techniques Used: Expressing, Immunohistochemistry, Labeling

    11) Product Images from "Daratumumab induces CD38 internalization and impairs myeloma cell adhesion"

    Article Title: Daratumumab induces CD38 internalization and impairs myeloma cell adhesion

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2018.1486948

    CD38 is internalized into CD38 + MM cells. (A) Schematic representation of surface and intracellular staining of CD38 in MM cell lines incubated with mouse anti-human CD38 Ab. Cells were stained with Anti-mouse Fc Alexa Fluor 568 for surface staining, washed, fixed, and permeabilized, and then stained with Anti-mouse Fc Alexa Fluor 633 for intracellular anti-human CD38 Ab/CD38 complex staining. ( B ) Flowsight cytometric analysis (using intracellular localization application wizard) showed CD38 internalization into different MM cell lines; internalization score was calculated by IDEAS Amnis software for anti-human CD38 antibody treated cells. The gating strategy to define the internalized and the surface AbCD38/CD38 populations is shown in Sup. Figure1. Internalization score was observed to be 39%, 28%, and 38% for NCI-H929, MM.1S and L363 cells, respectively, whereas internalization was only 1.5% for U266 cells, which do not express CD38. For replicates, ( C ) Bar diagram showing mean CD38 internalization score in MM.1S, L363, NCI-H929 cells but not in U266 cells with low CD38 expression. ( D ) Flowsight cytometric analysis showed CD38/Dara internalization into MM.1S cells treated with Dara. Secondary antibodies for surface and intracellular staining were FITC conjugated and TRITC conjugated anti-human IgG, respectively. Internalization score was similarly evaluated and observed to be 57% for MM.1S cells treated with Dara. Anti-HER2 humanized antibody (Trastuzumab) was used as a control and not observed to be internalized into MM.1S cells (erb2 negative). ( E ) Representative images of treated cells; for improved visualization, green (FITC) and red (TRITC) colors were assigned in IDEAS software to indicate surface staining and intracellular staining, respectively. Brightfield (BF), internalization (Merged) indicate single cell with internalized CD38/Dara.
    Figure Legend Snippet: CD38 is internalized into CD38 + MM cells. (A) Schematic representation of surface and intracellular staining of CD38 in MM cell lines incubated with mouse anti-human CD38 Ab. Cells were stained with Anti-mouse Fc Alexa Fluor 568 for surface staining, washed, fixed, and permeabilized, and then stained with Anti-mouse Fc Alexa Fluor 633 for intracellular anti-human CD38 Ab/CD38 complex staining. ( B ) Flowsight cytometric analysis (using intracellular localization application wizard) showed CD38 internalization into different MM cell lines; internalization score was calculated by IDEAS Amnis software for anti-human CD38 antibody treated cells. The gating strategy to define the internalized and the surface AbCD38/CD38 populations is shown in Sup. Figure1. Internalization score was observed to be 39%, 28%, and 38% for NCI-H929, MM.1S and L363 cells, respectively, whereas internalization was only 1.5% for U266 cells, which do not express CD38. For replicates, ( C ) Bar diagram showing mean CD38 internalization score in MM.1S, L363, NCI-H929 cells but not in U266 cells with low CD38 expression. ( D ) Flowsight cytometric analysis showed CD38/Dara internalization into MM.1S cells treated with Dara. Secondary antibodies for surface and intracellular staining were FITC conjugated and TRITC conjugated anti-human IgG, respectively. Internalization score was similarly evaluated and observed to be 57% for MM.1S cells treated with Dara. Anti-HER2 humanized antibody (Trastuzumab) was used as a control and not observed to be internalized into MM.1S cells (erb2 negative). ( E ) Representative images of treated cells; for improved visualization, green (FITC) and red (TRITC) colors were assigned in IDEAS software to indicate surface staining and intracellular staining, respectively. Brightfield (BF), internalization (Merged) indicate single cell with internalized CD38/Dara.

    Techniques Used: Staining, Incubation, Software, Expressing

    12) Product Images from "Daratumumab induces CD38 internalization and impairs myeloma cell adhesion"

    Article Title: Daratumumab induces CD38 internalization and impairs myeloma cell adhesion

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2018.1486948

    CD38 is internalized into CD38 + MM cells. (A) Schematic representation of surface and intracellular staining of CD38 in MM cell lines incubated with mouse anti-human CD38 Ab. Cells were stained with Anti-mouse Fc Alexa Fluor 568 for surface staining, washed, fixed, and permeabilized, and then stained with Anti-mouse Fc Alexa Fluor 633 for intracellular anti-human CD38 Ab/CD38 complex staining. ( B ) Flowsight cytometric analysis (using intracellular localization application wizard) showed CD38 internalization into different MM cell lines; internalization score was calculated by IDEAS Amnis software for anti-human CD38 antibody treated cells. The gating strategy to define the internalized and the surface AbCD38/CD38 populations is shown in Sup. Figure1. Internalization score was observed to be 39%, 28%, and 38% for NCI-H929, MM.1S and L363 cells, respectively, whereas internalization was only 1.5% for U266 cells, which do not express CD38. For replicates, ( C ) Bar diagram showing mean CD38 internalization score in MM.1S, L363, NCI-H929 cells but not in U266 cells with low CD38 expression. ( D ) Flowsight cytometric analysis showed CD38/Dara internalization into MM.1S cells treated with Dara. Secondary antibodies for surface and intracellular staining were FITC conjugated and TRITC conjugated anti-human IgG, respectively. Internalization score was similarly evaluated and observed to be 57% for MM.1S cells treated with Dara. Anti-HER2 humanized antibody (Trastuzumab) was used as a control and not observed to be internalized into MM.1S cells (erb2 negative). ( E ) Representative images of treated cells; for improved visualization, green (FITC) and red (TRITC) colors were assigned in IDEAS software to indicate surface staining and intracellular staining, respectively. Brightfield (BF), internalization (Merged) indicate single cell with internalized CD38/Dara.
    Figure Legend Snippet: CD38 is internalized into CD38 + MM cells. (A) Schematic representation of surface and intracellular staining of CD38 in MM cell lines incubated with mouse anti-human CD38 Ab. Cells were stained with Anti-mouse Fc Alexa Fluor 568 for surface staining, washed, fixed, and permeabilized, and then stained with Anti-mouse Fc Alexa Fluor 633 for intracellular anti-human CD38 Ab/CD38 complex staining. ( B ) Flowsight cytometric analysis (using intracellular localization application wizard) showed CD38 internalization into different MM cell lines; internalization score was calculated by IDEAS Amnis software for anti-human CD38 antibody treated cells. The gating strategy to define the internalized and the surface AbCD38/CD38 populations is shown in Sup. Figure1. Internalization score was observed to be 39%, 28%, and 38% for NCI-H929, MM.1S and L363 cells, respectively, whereas internalization was only 1.5% for U266 cells, which do not express CD38. For replicates, ( C ) Bar diagram showing mean CD38 internalization score in MM.1S, L363, NCI-H929 cells but not in U266 cells with low CD38 expression. ( D ) Flowsight cytometric analysis showed CD38/Dara internalization into MM.1S cells treated with Dara. Secondary antibodies for surface and intracellular staining were FITC conjugated and TRITC conjugated anti-human IgG, respectively. Internalization score was similarly evaluated and observed to be 57% for MM.1S cells treated with Dara. Anti-HER2 humanized antibody (Trastuzumab) was used as a control and not observed to be internalized into MM.1S cells (erb2 negative). ( E ) Representative images of treated cells; for improved visualization, green (FITC) and red (TRITC) colors were assigned in IDEAS software to indicate surface staining and intracellular staining, respectively. Brightfield (BF), internalization (Merged) indicate single cell with internalized CD38/Dara.

    Techniques Used: Staining, Incubation, Software, Expressing

    13) Product Images from "Non-peptidergic primary afferents are presynaptic to neurokinin-1 receptor immunoreactive lamina I projection neurons in rat spinal cord"

    Article Title: Non-peptidergic primary afferents are presynaptic to neurokinin-1 receptor immunoreactive lamina I projection neurons in rat spinal cord

    Journal: Molecular Pain

    doi: 10.1186/1744-8069-8-64

    Example of a quadruple labeling observed at the confocal level using a multi-track approach. In this image the following signals were simultaneously detected: CGRP (green); IB4 binding (red); CTb transported retrogradely from the parabrachial nucleus (blue); NK-1r (white). A fusiform neuron, double labeled with CTb and NK-1r, is innervated by CGRP-IR boutons (arrowhead) and IB4+ (arrow) boutons, which represent distinct populations. However, a small population of varicosities co-labeled for CGRP and IB4 (curved arrow) was detected. Scale bar ( A-E ) = 20 μm.
    Figure Legend Snippet: Example of a quadruple labeling observed at the confocal level using a multi-track approach. In this image the following signals were simultaneously detected: CGRP (green); IB4 binding (red); CTb transported retrogradely from the parabrachial nucleus (blue); NK-1r (white). A fusiform neuron, double labeled with CTb and NK-1r, is innervated by CGRP-IR boutons (arrowhead) and IB4+ (arrow) boutons, which represent distinct populations. However, a small population of varicosities co-labeled for CGRP and IB4 (curved arrow) was detected. Scale bar ( A-E ) = 20 μm.

    Techniques Used: Labeling, Binding Assay, CtB Assay

    Confocal images at high magnification obtained from parasagittal spinal cord sections showing CGRP-IR (green) and P2X3-IR (red) varicosities in the superficial dorsal horn. P2X3-IR varicosities were present in considerable number in lamina I (LI) but were more highly concentrated in inner lamina II (LIIi). Scale bar = 20 μm.
    Figure Legend Snippet: Confocal images at high magnification obtained from parasagittal spinal cord sections showing CGRP-IR (green) and P2X3-IR (red) varicosities in the superficial dorsal horn. P2X3-IR varicosities were present in considerable number in lamina I (LI) but were more highly concentrated in inner lamina II (LIIi). Scale bar = 20 μm.

    Techniques Used:

    Confocal images at high power obtained from horizontal spinal cord sections. In a confocal optical section from lamina I adjacent to the white matter ( A ), note the relatively abundant P2X3-IR fibers with varicosities (boutons). CGRP-IR fibers and boutons were considerably more abundant in this lamina. In a confocal optical section from inner lamina II ( B ), note the very high density of P2X3-IR fibers and varicosities, higher than that of CGRP-IR fibers in lamina I. Note that most varicosities display either P2X3 or CGRP immunoreactivity, although some co-localization is observed (yellow). Scale bar ( A, B ) = 20 μm.
    Figure Legend Snippet: Confocal images at high power obtained from horizontal spinal cord sections. In a confocal optical section from lamina I adjacent to the white matter ( A ), note the relatively abundant P2X3-IR fibers with varicosities (boutons). CGRP-IR fibers and boutons were considerably more abundant in this lamina. In a confocal optical section from inner lamina II ( B ), note the very high density of P2X3-IR fibers and varicosities, higher than that of CGRP-IR fibers in lamina I. Note that most varicosities display either P2X3 or CGRP immunoreactivity, although some co-localization is observed (yellow). Scale bar ( A, B ) = 20 μm.

    Techniques Used:

    CGRP, IB4 and P2X3 staining in transverse spinal cord sections. A and B show low magnification confocal images of CGRP-IR and IB4 positive (A) or P2X3-IR (B) fibers. C and D represent high magnification confocal images from the middle third of the latero-medial extent of the superficial dorsal horn. In C , note that there is limited co-localization of IB4 and CGRP (in yellow). Arrowheads show axonal varicosities (boutons) from non-peptidergic fibers in lamina I, which do not co-localize CGRP immunoreactivity. The framed regions in A and B indicate the approximate regions from where C and D , respectively, were obtained (the latter originate from other sections). CGRP (in green); IB4 (in red); P2X3 (in red). Scale bar ( A , B ) = 200 μm; scale bar ( C , D ) = 20 μm.
    Figure Legend Snippet: CGRP, IB4 and P2X3 staining in transverse spinal cord sections. A and B show low magnification confocal images of CGRP-IR and IB4 positive (A) or P2X3-IR (B) fibers. C and D represent high magnification confocal images from the middle third of the latero-medial extent of the superficial dorsal horn. In C , note that there is limited co-localization of IB4 and CGRP (in yellow). Arrowheads show axonal varicosities (boutons) from non-peptidergic fibers in lamina I, which do not co-localize CGRP immunoreactivity. The framed regions in A and B indicate the approximate regions from where C and D , respectively, were obtained (the latter originate from other sections). CGRP (in green); IB4 (in red); P2X3 (in red). Scale bar ( A , B ) = 200 μm; scale bar ( C , D ) = 20 μm.

    Techniques Used: Staining

    14) Product Images from "The Mi-2/NuRD complex associates with pericentromeric heterochromatin during S phase in rapidly proliferating lymphoid cells"

    Article Title: The Mi-2/NuRD complex associates with pericentromeric heterochromatin during S phase in rapidly proliferating lymphoid cells

    Journal: Chromosoma

    doi: 10.1007/s00412-009-0207-7

    Mi-2/NuRD enrichment at pericentromeric heterochromatin is tightly linked to DNA replication and chromatin assembly A) Graph shows the proportion of cells with NuRD bodies in each stage of the cell cycle, on the basis of BrdU incorporation and CENP-F staining. Cells containing NuRD bodies were identified and scored as to whether they were in S, G1 or G2 phase. Overall, 51% of cells in the asynchronous culture were scored as being in S phase, irrespective of the presence of NuRD bodies. B) Graph shows the proportion of synchronized cells that contain NuRD bodies. Cells were synchronized at the G1/S border, released into S phase, and analyzed at several time points (unsynchronized, 0 hrs, 1.5 hrs, 3 hrs, 4.5 hrs, 6 hrs and 8 hrs after release). Cells were scored as to whether Mi-2/NuRD foci were observed. Left side of chart shows DNA content analysis after flow sorting. C) Ramos cells were pulsed for 15 minutes with BrdU prior to cytocentrifugation, fixation and detection with MTA3 (red), BrdU (green) and PCNA (AlexaFluor 647, purple) antibodies. The lower panel shows an expanded view of two brightly staining foci in the nucleus pictured in the upper panel. Scale bar indicates 5 μm. D) Ramos cells were cytocentrifuged and stained with MTA3 (directly labeled with AlexaFluor 568) and CAF-1 p150 (detected with AlexaFluor 488 secondary antibodies). Scale bar indicates 5 μm. E. Ramos cells were cytocentrifuged and immunostained with antibodies against MTA3 (green) and HP1g (red). Images were acquired using confocal microscopy with subsequent deconvolution. Panel shows an expanded view of one NuRD body. The image presented is a summed Z-series. Scale bar indicates 2.5 μm.
    Figure Legend Snippet: Mi-2/NuRD enrichment at pericentromeric heterochromatin is tightly linked to DNA replication and chromatin assembly A) Graph shows the proportion of cells with NuRD bodies in each stage of the cell cycle, on the basis of BrdU incorporation and CENP-F staining. Cells containing NuRD bodies were identified and scored as to whether they were in S, G1 or G2 phase. Overall, 51% of cells in the asynchronous culture were scored as being in S phase, irrespective of the presence of NuRD bodies. B) Graph shows the proportion of synchronized cells that contain NuRD bodies. Cells were synchronized at the G1/S border, released into S phase, and analyzed at several time points (unsynchronized, 0 hrs, 1.5 hrs, 3 hrs, 4.5 hrs, 6 hrs and 8 hrs after release). Cells were scored as to whether Mi-2/NuRD foci were observed. Left side of chart shows DNA content analysis after flow sorting. C) Ramos cells were pulsed for 15 minutes with BrdU prior to cytocentrifugation, fixation and detection with MTA3 (red), BrdU (green) and PCNA (AlexaFluor 647, purple) antibodies. The lower panel shows an expanded view of two brightly staining foci in the nucleus pictured in the upper panel. Scale bar indicates 5 μm. D) Ramos cells were cytocentrifuged and stained with MTA3 (directly labeled with AlexaFluor 568) and CAF-1 p150 (detected with AlexaFluor 488 secondary antibodies). Scale bar indicates 5 μm. E. Ramos cells were cytocentrifuged and immunostained with antibodies against MTA3 (green) and HP1g (red). Images were acquired using confocal microscopy with subsequent deconvolution. Panel shows an expanded view of one NuRD body. The image presented is a summed Z-series. Scale bar indicates 2.5 μm.

    Techniques Used: BrdU Incorporation Assay, Staining, Flow Cytometry, Labeling, Confocal Microscopy

    Mi-2/NuRD subunits are enriched in nuclear foci A) Ramos and H929 cells were cytocentrifuged and immunostained with antibodies against MTA3 (left) or Mi-2 (right). Primary antibodies were detected with AlexaFluor 488 conjugated secondary antibodies (green). Cells are counterstained with DAPI (blue). Scale bar (top left corner) indicates 5 μm. B) Two color immunofluorescence in cytocentrifuged Ramos cells. In the top panel, Mi-2 was detected with AlexaFluor 568 secondary antibody; the MTA3 antibody was directly labeled with AlexaFluor 488. In the lower panel, MTA3 and BCL6 were detected with AlexaFluor 488 (green) and AlexaFluor568 (red) secondary antibodies. Scale bar indicates 5 μm. C) Cytocentrifuged Ramos cells were immunostained with antibodies against other members of the Mi-2/NuRD complex (MBD2, HDAC1, HDAC2, Mi-2a and Mi-2b), and AlexaFluor 488 or 568 secondary antibodies. Cells were counterstained with DAPI. Scale bar indicates 5 μm.
    Figure Legend Snippet: Mi-2/NuRD subunits are enriched in nuclear foci A) Ramos and H929 cells were cytocentrifuged and immunostained with antibodies against MTA3 (left) or Mi-2 (right). Primary antibodies were detected with AlexaFluor 488 conjugated secondary antibodies (green). Cells are counterstained with DAPI (blue). Scale bar (top left corner) indicates 5 μm. B) Two color immunofluorescence in cytocentrifuged Ramos cells. In the top panel, Mi-2 was detected with AlexaFluor 568 secondary antibody; the MTA3 antibody was directly labeled with AlexaFluor 488. In the lower panel, MTA3 and BCL6 were detected with AlexaFluor 488 (green) and AlexaFluor568 (red) secondary antibodies. Scale bar indicates 5 μm. C) Cytocentrifuged Ramos cells were immunostained with antibodies against other members of the Mi-2/NuRD complex (MBD2, HDAC1, HDAC2, Mi-2a and Mi-2b), and AlexaFluor 488 or 568 secondary antibodies. Cells were counterstained with DAPI. Scale bar indicates 5 μm.

    Techniques Used: Immunofluorescence, Labeling

    15) Product Images from "Hoxb8 Intersection Defines a Role for Lmx1b in Excitatory Dorsal Horn Neuron Development, Spinofugal Connectivity, and Nociception"

    Article Title: Hoxb8 Intersection Defines a Role for Lmx1b in Excitatory Dorsal Horn Neuron Development, Spinofugal Connectivity, and Nociception

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4690-14.2015

    Abnormal innervation of dorsal horn by primary afferents that are required for mechanical sensation. A , D , Schematic illustration of the innervation of the dorsal horn by CGRP-positive primary afferents (red) that relay thermal sensation and IB4-positive afferents (green) relaying mechanical pain sensation. B , E , Stainings with an antibody against CGRP and with the isolectin IB4 in adult Lmxb CTL ( B ) and Lmx1b CND ( E ) spinal cord at cervical level C7, area of dorsal horn as in A , D (inbox). CGRP-positive fibers innervate lamina I and outer lamina II in Lmx1b CTL and Lmx1b CND mice. IB4-positive afferents innervate the outer lamina II in Lmx1b CTL spinal cord ( B ). In Lmx1b CND mice, however, the innervated area in the dorsal horn is not as dense and shows IB4-positive aggregates ( E , arrowheads). C , F , Labeling of PKCγ and IB4 in adult Lmx1b CTL ( C ) spinal cord at cervical level C7 labels PKCγ-positive cells in inner lamina II of the dorsal horn. In Lmx1b CND dorsal horn, the expression area of PKCγ and the termination area of IB4-positive afferents overlap and occupy a more superficial area of the dorsal horn ( F ). G , J , Immunostaining with antibodies against CGRP and NK1R shows innervation of NK1R-expressing neurons in lamina I of the dorsal horn by CGRP-expressing primary afferents in Lmx1b CTL mice ( G ). Lmx1b CND mice show normal innervation of the dorsal horn by CGRP-expressing afferents but absence of NK1R-expressing cells in the most superficial lamina ( J ). H–L , Immunodetection of synapsin and staining with IB4 at cervical level C7 in adult Lmx1b CTL and Lmx1b CND mice. The synaptic protein synapsin labels many synapses in the innervation area of IB4-positive afferents in the dorsal horn of both Lmx1b CTL and Lmx1b CND mice. M , N , Quantification of synapsin + and IB4 + puncta in the innervation area of IB4-positive afferents, assessed in an area of the size shown in I and L in the dorsal horn, shows no significant difference between Lmx1b CTL and Lmx1b CDN mice. Also, the overlap of synapsin and IB4-positive puncta is unchanged in the analyzed area (557.5 ± 41.8 synapsin + puncta in analyzed area in Lmx1b CTL vs 501.3 ± 17.5 in Lmx1b CND , p = 0.1; 369 ± 34.8 IB4 + puncta in Lmx1b CTL vs 239.9 ± 80.3 in Lmx1b CND , p = 0.06; 57.2 ± 42.1 synapsin + /IB4 + puncta in Lmx1b CTL vs 53 ± 10.7 in Lmx1b CND , p = 0.88; n = 3). Scale bars: B , C , E–H , J , K , 20 μm; I , L , 10 μm. ns, Not significant.
    Figure Legend Snippet: Abnormal innervation of dorsal horn by primary afferents that are required for mechanical sensation. A , D , Schematic illustration of the innervation of the dorsal horn by CGRP-positive primary afferents (red) that relay thermal sensation and IB4-positive afferents (green) relaying mechanical pain sensation. B , E , Stainings with an antibody against CGRP and with the isolectin IB4 in adult Lmxb CTL ( B ) and Lmx1b CND ( E ) spinal cord at cervical level C7, area of dorsal horn as in A , D (inbox). CGRP-positive fibers innervate lamina I and outer lamina II in Lmx1b CTL and Lmx1b CND mice. IB4-positive afferents innervate the outer lamina II in Lmx1b CTL spinal cord ( B ). In Lmx1b CND mice, however, the innervated area in the dorsal horn is not as dense and shows IB4-positive aggregates ( E , arrowheads). C , F , Labeling of PKCγ and IB4 in adult Lmx1b CTL ( C ) spinal cord at cervical level C7 labels PKCγ-positive cells in inner lamina II of the dorsal horn. In Lmx1b CND dorsal horn, the expression area of PKCγ and the termination area of IB4-positive afferents overlap and occupy a more superficial area of the dorsal horn ( F ). G , J , Immunostaining with antibodies against CGRP and NK1R shows innervation of NK1R-expressing neurons in lamina I of the dorsal horn by CGRP-expressing primary afferents in Lmx1b CTL mice ( G ). Lmx1b CND mice show normal innervation of the dorsal horn by CGRP-expressing afferents but absence of NK1R-expressing cells in the most superficial lamina ( J ). H–L , Immunodetection of synapsin and staining with IB4 at cervical level C7 in adult Lmx1b CTL and Lmx1b CND mice. The synaptic protein synapsin labels many synapses in the innervation area of IB4-positive afferents in the dorsal horn of both Lmx1b CTL and Lmx1b CND mice. M , N , Quantification of synapsin + and IB4 + puncta in the innervation area of IB4-positive afferents, assessed in an area of the size shown in I and L in the dorsal horn, shows no significant difference between Lmx1b CTL and Lmx1b CDN mice. Also, the overlap of synapsin and IB4-positive puncta is unchanged in the analyzed area (557.5 ± 41.8 synapsin + puncta in analyzed area in Lmx1b CTL vs 501.3 ± 17.5 in Lmx1b CND , p = 0.1; 369 ± 34.8 IB4 + puncta in Lmx1b CTL vs 239.9 ± 80.3 in Lmx1b CND , p = 0.06; 57.2 ± 42.1 synapsin + /IB4 + puncta in Lmx1b CTL vs 53 ± 10.7 in Lmx1b CND , p = 0.88; n = 3). Scale bars: B , C , E–H , J , K , 20 μm; I , L , 10 μm. ns, Not significant.

    Techniques Used: CTL Assay, Mouse Assay, Labeling, Expressing, Immunostaining, Immunodetection, Staining

    16) Product Images from "Activation-induced deaminase cloning, localization, and protein extraction from young VH-mutant rabbit appendix"

    Article Title: Activation-induced deaminase cloning, localization, and protein extraction from young VH-mutant rabbit appendix

    Journal:

    doi: 10.1073/pnas.0501338102

    Seven-micrometer 4.5-wk-old ali / ali appendix sections were stained with Alexa Fluor 568-anti-RAID (red), FITC-anti-IgM (green), Alexa Fluor 647-anti-macrophage (white), and DAPI (blue). ( A ) Whole follicle. ( B – D ) High-magnification images captured
    Figure Legend Snippet: Seven-micrometer 4.5-wk-old ali / ali appendix sections were stained with Alexa Fluor 568-anti-RAID (red), FITC-anti-IgM (green), Alexa Fluor 647-anti-macrophage (white), and DAPI (blue). ( A ) Whole follicle. ( B – D ) High-magnification images captured

    Techniques Used: Staining

    17) Product Images from "Porphyromonas gingivalis induces penetration of lipopolysaccharide and peptidoglycan through the gingival epithelium via degradation of junctional adhesion molecule 1"

    Article Title: Porphyromonas gingivalis induces penetration of lipopolysaccharide and peptidoglycan through the gingival epithelium via degradation of junctional adhesion molecule 1

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008124

    P . gingivalis degrades JAM1 of gingival epithelium, causing penetration of LPS and PGN. (A, B) Schematic illustration of the three-dimensional culture (A) and confocal microscopic cross-sectional images (B) of the three-dimensional culture of IHGE cells. Gingival epithelial tissues (WT or overexpressing JAM1) were infected with P . gingivalis for 30 min. Tissues were then fixed, stained with anti-JAM1 (white) and Alexa Fluor 568–conjugated phalloidin (magenta), and analyzed by confocal microscopy. Scale bars, 30 μm. (C – G) Permeability to 40 kDa FITC-dextran (C), FITC– P . gingivalis LPS (D), FITC– P . gingivalis PGN (E), FITC– E . coli LPS (F), and FITC– S . aureus PGN (G) of gingival epithelial tissues (WT or overexpressing JAM1) infected with P . gingivalis . Three-dimensional tissues on culture inserts were infected with P . gingivalis and FITC-labeled tracer in the upper compartment. Following 30 min of incubation, the transmission of tracer from the upper compartment to the lower compartment was analyzed by spectrometry. Results are expressed as fold change relative to uninfected WT cells and are the means ± SD of seven technical replicates. *, p
    Figure Legend Snippet: P . gingivalis degrades JAM1 of gingival epithelium, causing penetration of LPS and PGN. (A, B) Schematic illustration of the three-dimensional culture (A) and confocal microscopic cross-sectional images (B) of the three-dimensional culture of IHGE cells. Gingival epithelial tissues (WT or overexpressing JAM1) were infected with P . gingivalis for 30 min. Tissues were then fixed, stained with anti-JAM1 (white) and Alexa Fluor 568–conjugated phalloidin (magenta), and analyzed by confocal microscopy. Scale bars, 30 μm. (C – G) Permeability to 40 kDa FITC-dextran (C), FITC– P . gingivalis LPS (D), FITC– P . gingivalis PGN (E), FITC– E . coli LPS (F), and FITC– S . aureus PGN (G) of gingival epithelial tissues (WT or overexpressing JAM1) infected with P . gingivalis . Three-dimensional tissues on culture inserts were infected with P . gingivalis and FITC-labeled tracer in the upper compartment. Following 30 min of incubation, the transmission of tracer from the upper compartment to the lower compartment was analyzed by spectrometry. Results are expressed as fold change relative to uninfected WT cells and are the means ± SD of seven technical replicates. *, p

    Techniques Used: Infection, Staining, Confocal Microscopy, Permeability, Labeling, Incubation, Transmission Assay

    JAM1 is required for epithelial barrier function of gingival epithelial tissues. (A, B) Schematic illustration (A) and confocal microscopic cross-sectional images (B) of 3D-tissue model expressing shLuc or shJAM1. Gingival epithelial tissues were fixed, stained with anti-JAM1 (white) and Alexa Fluor 568–conjugated phalloidin (magenta), and analyzed by confocal microscopy. Scale bars, 30 μm. (C – G) Permeability to 40 kDa FITC–dextran (C), FITC– P . gingivalis LPS (D), FITC– P . gingivalis PGN (E), FITC– E . coli LPS (F), and FITC– S . aureus PGN (G) in gingival epithelial tissues expressing shLuc and shJAM1. Results are expressed as fold change relative to epithelium expressing shLuc and are the means ± SD of seven technical replicates. *, p
    Figure Legend Snippet: JAM1 is required for epithelial barrier function of gingival epithelial tissues. (A, B) Schematic illustration (A) and confocal microscopic cross-sectional images (B) of 3D-tissue model expressing shLuc or shJAM1. Gingival epithelial tissues were fixed, stained with anti-JAM1 (white) and Alexa Fluor 568–conjugated phalloidin (magenta), and analyzed by confocal microscopy. Scale bars, 30 μm. (C – G) Permeability to 40 kDa FITC–dextran (C), FITC– P . gingivalis LPS (D), FITC– P . gingivalis PGN (E), FITC– E . coli LPS (F), and FITC– S . aureus PGN (G) in gingival epithelial tissues expressing shLuc and shJAM1. Results are expressed as fold change relative to epithelium expressing shLuc and are the means ± SD of seven technical replicates. *, p

    Techniques Used: Expressing, Staining, Confocal Microscopy, Permeability

    Confocal microscopic images of JAM1 in IHGE cells infected with P . gingivalis WT or the Δ kgp Δ rgpA Δ rgpB mutant. (A) IHGE cells were infected with P . gingivalis WT or the Δ kgp Δ rgpA Δ rgpB mutant at an MOI of 100 for 1.5 h. The cells were then fixed, stained with DAPI (cyan) and anti-JAM1 (yellow), and analyzed by confocal microscopy. Scale bars, 10 μm. ( B, C) Schematic illustration (B) and confocal microscopic cross-sectional images (C) of the 3D-tissue model of IHGE cells. Gingival epithelial tissues on coverslips were infected with P . gingivalis WT or the Δ kgp Δ rgpA Δ rgpB mutant for 2 h. The tissues were then fixed, stained with anti-JAM1 (white) and Alexa Fluor 568–conjugated phalloidin (magenta), and analyzed by confocal microscopy. Scale bars, 30 μm.
    Figure Legend Snippet: Confocal microscopic images of JAM1 in IHGE cells infected with P . gingivalis WT or the Δ kgp Δ rgpA Δ rgpB mutant. (A) IHGE cells were infected with P . gingivalis WT or the Δ kgp Δ rgpA Δ rgpB mutant at an MOI of 100 for 1.5 h. The cells were then fixed, stained with DAPI (cyan) and anti-JAM1 (yellow), and analyzed by confocal microscopy. Scale bars, 10 μm. ( B, C) Schematic illustration (B) and confocal microscopic cross-sectional images (C) of the 3D-tissue model of IHGE cells. Gingival epithelial tissues on coverslips were infected with P . gingivalis WT or the Δ kgp Δ rgpA Δ rgpB mutant for 2 h. The tissues were then fixed, stained with anti-JAM1 (white) and Alexa Fluor 568–conjugated phalloidin (magenta), and analyzed by confocal microscopy. Scale bars, 30 μm.

    Techniques Used: Infection, Mutagenesis, Staining, Confocal Microscopy

    18) Product Images from "SIGIRR, a Negative Regulator of TLR/IL-1R Signalling Promotes Microbiota Dependent Resistance to Colonization by Enteric Bacterial Pathogens"

    Article Title: SIGIRR, a Negative Regulator of TLR/IL-1R Signalling Promotes Microbiota Dependent Resistance to Colonization by Enteric Bacterial Pathogens

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003539

    MyD88 signaling is required for the survival of infected Sigrr −/− mice. WT, Sigirr −/− , Myd88/Sigirr −/− , Tlr2/Sigirr −/− , and Tlr4/Sigirr −/− mice were infected by C. rodentium for 6 and 10 days. (A) Myd88/Sigirr −/− mice required euthanization by D8 pi whereas the other mouse groups survived the infection. (B) All mice on a Sigirr −/− background carried significantly heavier pathogen burdens and (C) showed more severe colitis compared to WT mice at D6 and D10 pi. (C–D) Immunostaining for the proliferation marker Ki-67 demonstrated infected Sigirr−/− , Tlr2/Sigirr −/− and Tlr4/Sigirr −/− display elevated IEC proliferation compared to WT mice. (E) phospho STAT-3 staining is restored in Tlr2/Sigirr −/− mice, while (F) the heightened barrier disruption seen in infected Tlr2−/− mice is normalized in Tlr2/Sigirr −/− mice. Pathogen counts represent mucosal associated bacteria. Results are pooled from 2–3 independent infections, each with n = 3–4 per group. Error bars = SEM, (Student t test (Figure B, D) and one way ANOVA (Figure D), *P
    Figure Legend Snippet: MyD88 signaling is required for the survival of infected Sigrr −/− mice. WT, Sigirr −/− , Myd88/Sigirr −/− , Tlr2/Sigirr −/− , and Tlr4/Sigirr −/− mice were infected by C. rodentium for 6 and 10 days. (A) Myd88/Sigirr −/− mice required euthanization by D8 pi whereas the other mouse groups survived the infection. (B) All mice on a Sigirr −/− background carried significantly heavier pathogen burdens and (C) showed more severe colitis compared to WT mice at D6 and D10 pi. (C–D) Immunostaining for the proliferation marker Ki-67 demonstrated infected Sigirr−/− , Tlr2/Sigirr −/− and Tlr4/Sigirr −/− display elevated IEC proliferation compared to WT mice. (E) phospho STAT-3 staining is restored in Tlr2/Sigirr −/− mice, while (F) the heightened barrier disruption seen in infected Tlr2−/− mice is normalized in Tlr2/Sigirr −/− mice. Pathogen counts represent mucosal associated bacteria. Results are pooled from 2–3 independent infections, each with n = 3–4 per group. Error bars = SEM, (Student t test (Figure B, D) and one way ANOVA (Figure D), *P

    Techniques Used: Infection, Mouse Assay, Immunostaining, Marker, Staining

    19) Product Images from "Immune Stimulation Using a Gut Microbe-Based Immunotherapy Reduces Disease Pathology and Improves Barrier Function in Ulcerative Colitis"

    Article Title: Immune Stimulation Using a Gut Microbe-Based Immunotherapy Reduces Disease Pathology and Improves Barrier Function in Ulcerative Colitis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02211

    QBECO treatment reduced disease severity in Muc-2 −/− mice. Muc2 −/− mice were injected subcutaneously with placebo or QBECO every other day for 30 days and colonic tissue was collected for histology grading (A,B) . Colonic tissues were also stained with (C) anti-Ly6G to assess neutrophil number and (D) anti-CD3 to assess the number of T cells in colonic tissues. Control n = 15 QBECO: n = 10, mean ± SEM, * p
    Figure Legend Snippet: QBECO treatment reduced disease severity in Muc-2 −/− mice. Muc2 −/− mice were injected subcutaneously with placebo or QBECO every other day for 30 days and colonic tissue was collected for histology grading (A,B) . Colonic tissues were also stained with (C) anti-Ly6G to assess neutrophil number and (D) anti-CD3 to assess the number of T cells in colonic tissues. Control n = 15 QBECO: n = 10, mean ± SEM, * p

    Techniques Used: Mouse Assay, Injection, Staining

    20) Product Images from "Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks"

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    Journal: bioRxiv

    doi: 10.1101/864769

    Twinfilin uncaps filament barbed ends in vitro. (A) A schematic overview on the in vitro single filament experimental approach. Actin filaments (10% AlexaFluor-488 labelled G-actin) were polymerized from spectrin-actin seeds and subsequently capped with 100 nM capping protein. Observation was done under constant microfluidics flow of either buffer alone or together with TWF-1 and/or V1 for 60 individual filaments in each condition. (B) Fraction of capped barbed ends over time with different concentrations of TWF-1. (C) Fraction of capped barbed ends over time either without or with 2.7 μ M TWF-1 and/or 37 μ M V1 protein.
    Figure Legend Snippet: Twinfilin uncaps filament barbed ends in vitro. (A) A schematic overview on the in vitro single filament experimental approach. Actin filaments (10% AlexaFluor-488 labelled G-actin) were polymerized from spectrin-actin seeds and subsequently capped with 100 nM capping protein. Observation was done under constant microfluidics flow of either buffer alone or together with TWF-1 and/or V1 for 60 individual filaments in each condition. (B) Fraction of capped barbed ends over time with different concentrations of TWF-1. (C) Fraction of capped barbed ends over time either without or with 2.7 μ M TWF-1 and/or 37 μ M V1 protein.

    Techniques Used: In Vitro

    21) Product Images from "Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks"

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    Journal: bioRxiv

    doi: 10.1101/864769

    Expression of EGFP-TWF-1 rescues the twf1/twf2 knockout phenotype. Representative images of twf1/twf2-KO cells expressing EGFP-TWF-1 and stained with AlexaFluor-647 phalloidin and DAPI to visualize F-actin and nuclei, respectively. Scale bars = 20 μ m.
    Figure Legend Snippet: Expression of EGFP-TWF-1 rescues the twf1/twf2 knockout phenotype. Representative images of twf1/twf2-KO cells expressing EGFP-TWF-1 and stained with AlexaFluor-647 phalloidin and DAPI to visualize F-actin and nuclei, respectively. Scale bars = 20 μ m.

    Techniques Used: Expressing, Knock-Out, Staining

    Twinfilin-1/twinfilin-2 knockout leads to an accumulation of F-actin on endosomes at the perinuclear region. (A) Phalloidin staining of wild-type B16-F1, and (B) twf1/twf2-KO cells after 7.5 min uptake of 20 μ g/ml AlexaFluor-647 transferrin. Scale bar = 20 μ m. The dotted square indicates the perinuclear region magnified in the insert. (C) Mean F-actin intensities in transferrin-positive endosomes of wild-type and twf1/twf2-KO cells as measured from AlexaFluor-555 phalloidin stained cells after 7.5 min intake of 20 μ g/ml AlexaFluor-647 phalloidin. Numbers of measured cells were: B16-F1 wt = 2,518, twf1/2-KO-g3 = 3,637, twf1/2-KO-g3 + EGFP-TWF1 = 158. Statistical significances were calculated with Mann-Whitney two-tailed test. ****, p
    Figure Legend Snippet: Twinfilin-1/twinfilin-2 knockout leads to an accumulation of F-actin on endosomes at the perinuclear region. (A) Phalloidin staining of wild-type B16-F1, and (B) twf1/twf2-KO cells after 7.5 min uptake of 20 μ g/ml AlexaFluor-647 transferrin. Scale bar = 20 μ m. The dotted square indicates the perinuclear region magnified in the insert. (C) Mean F-actin intensities in transferrin-positive endosomes of wild-type and twf1/twf2-KO cells as measured from AlexaFluor-555 phalloidin stained cells after 7.5 min intake of 20 μ g/ml AlexaFluor-647 phalloidin. Numbers of measured cells were: B16-F1 wt = 2,518, twf1/2-KO-g3 = 3,637, twf1/2-KO-g3 + EGFP-TWF1 = 158. Statistical significances were calculated with Mann-Whitney two-tailed test. ****, p

    Techniques Used: Knock-Out, Staining, MANN-WHITNEY, Two Tailed Test

    22) Product Images from "Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks"

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    Journal: bioRxiv

    doi: 10.1101/864769

    Twinfilin regulates capping protein localization and dynamics. (A) Localization of EGFP-CP in wild-type and twf1/twf2-KO B16-F1 cells, where F-actin was visualized with AlexaFluor-568 phalloidin. Panels in the middle and right are magnifications of lamellipodial regions highlighted in the whole cell images in left. Scale bars = 10 μ m. (B) Examples of line profiles generated across the center of lamellipodia as indicated with dotted lines. Data represent mean of 5 measurements of individual lamellipodia, with standard deviations shown. The ‘0 μ m’ value in x-axis is set to correspond the peak intensity of phalloidin. (C) The ratio of CP and F-actin co-localization widths were detected by measuring the width of localization at 50% of maximum intensity. Data points represent measurements from individual lamellipodia with mean values and standard deviations shown. Statistical significance was calculated with Student’s unpaired, two-tailed t-test. ****, p
    Figure Legend Snippet: Twinfilin regulates capping protein localization and dynamics. (A) Localization of EGFP-CP in wild-type and twf1/twf2-KO B16-F1 cells, where F-actin was visualized with AlexaFluor-568 phalloidin. Panels in the middle and right are magnifications of lamellipodial regions highlighted in the whole cell images in left. Scale bars = 10 μ m. (B) Examples of line profiles generated across the center of lamellipodia as indicated with dotted lines. Data represent mean of 5 measurements of individual lamellipodia, with standard deviations shown. The ‘0 μ m’ value in x-axis is set to correspond the peak intensity of phalloidin. (C) The ratio of CP and F-actin co-localization widths were detected by measuring the width of localization at 50% of maximum intensity. Data points represent measurements from individual lamellipodia with mean values and standard deviations shown. Statistical significance was calculated with Student’s unpaired, two-tailed t-test. ****, p

    Techniques Used: Generated, Two Tailed Test

    Examples of high-content and lamellipodia protrusion analysis. (A) Representative images of segmentation procedure in high-content image analysis. Wild-type and twinfilin-deficient B16-F1 cells were stained with DAPI and CellMask Deep Red to segment nuclei (outlined with blue) and cytoplasm (outlined with yellow). The Arp2/3-complex positive structures were detected with anti-p34 antibody staining and used as a mask for the Arp2/3-complex positive F-actin structures (the most-right panel). F-actin was stained with AlexaFluor-568 phalloidin. Cells touching the border of images were excluded from analysis. (B) A representative example of twf1/twf2-KO cell migrating on laminin coated glass imaged with DIC. Direction of migration is indicated with an arrow and kymographs were generated with line drawn across the lamellipodium as indicated with dotted red line. (C) Representative examples of kymographs generated from DIC time-lapse images of wild-type, twf1/twf2 knockout, and EGFP-TWF-1 rescue cells. Protrusions velocities were measured from the overall cell front protrusion as indicated with dotted red lines.
    Figure Legend Snippet: Examples of high-content and lamellipodia protrusion analysis. (A) Representative images of segmentation procedure in high-content image analysis. Wild-type and twinfilin-deficient B16-F1 cells were stained with DAPI and CellMask Deep Red to segment nuclei (outlined with blue) and cytoplasm (outlined with yellow). The Arp2/3-complex positive structures were detected with anti-p34 antibody staining and used as a mask for the Arp2/3-complex positive F-actin structures (the most-right panel). F-actin was stained with AlexaFluor-568 phalloidin. Cells touching the border of images were excluded from analysis. (B) A representative example of twf1/twf2-KO cell migrating on laminin coated glass imaged with DIC. Direction of migration is indicated with an arrow and kymographs were generated with line drawn across the lamellipodium as indicated with dotted red line. (C) Representative examples of kymographs generated from DIC time-lapse images of wild-type, twf1/twf2 knockout, and EGFP-TWF-1 rescue cells. Protrusions velocities were measured from the overall cell front protrusion as indicated with dotted red lines.

    Techniques Used: Staining, Migration, Generated, Knock-Out

    Representative images of wild-type and twinfilin knockout B16-F1 cells. (A) B16-F1 cells were stained with AlexaFluor-568 phalloidin (F-actin) and anti-p34 antibody (the Arp2/3 complex). Scale bars = 10 μ M. (B) Mean F-actin intensity in B16-F1 wild-type and twinfilin knockout cells. Number of measured cells were: B16-F1 wt = 1,958, twf1-KO-g1 = 1,045, twf2-KO-g3#1 = 1,285, twf1/2-KO-g3 = 1,265, twf1/2-KO-g4 = 1,707. (C) Mean F-actin intensity in the Arp2/3 complex positive regions of B16-F1 wild-type and knockout cells. The Arp2/3 complex positive regions were identified based on p34-antibody staining. Number of measured cells were: B16-F1 wt = 1,658, twf1-KO-g1 = 884, twf2-KO-g3#1 = 1,009, twf1/2-KO-g3 = 1,100, twf1/2-KO-g4 = 1157. Statistical significances in panels B and C were calculated with Mann-Whitney two-tailed test. ****, p
    Figure Legend Snippet: Representative images of wild-type and twinfilin knockout B16-F1 cells. (A) B16-F1 cells were stained with AlexaFluor-568 phalloidin (F-actin) and anti-p34 antibody (the Arp2/3 complex). Scale bars = 10 μ M. (B) Mean F-actin intensity in B16-F1 wild-type and twinfilin knockout cells. Number of measured cells were: B16-F1 wt = 1,958, twf1-KO-g1 = 1,045, twf2-KO-g3#1 = 1,285, twf1/2-KO-g3 = 1,265, twf1/2-KO-g4 = 1,707. (C) Mean F-actin intensity in the Arp2/3 complex positive regions of B16-F1 wild-type and knockout cells. The Arp2/3 complex positive regions were identified based on p34-antibody staining. Number of measured cells were: B16-F1 wt = 1,658, twf1-KO-g1 = 884, twf2-KO-g3#1 = 1,009, twf1/2-KO-g3 = 1,100, twf1/2-KO-g4 = 1157. Statistical significances in panels B and C were calculated with Mann-Whitney two-tailed test. ****, p

    Techniques Used: Knock-Out, Staining, MANN-WHITNEY, Two Tailed Test

    Knockout of twinfilins leads to abnormal F-actin accumulation in lamellipodia and perinuclear region. (A) Representative images of wild-type and twf1/twf2-KO mouse B16-F1 cells stained with AlexaFluor-568 phalloidin and anti-p34 antibody to visualize F-actin and the Arp2/3 complex, respectively. Scale bar = 10 μ m. (B) F-actin intensities in the cytoplasmic regions of wild-type, twf1/twf2-KO, and knockout cells expressing EGFP-TWF-1 measured by high-content image analysis. Number of cells analyzed were: B16-F1 wt = 4,875, twf1/twf2-KO-g3 = 5,731, twf1/twf2-KO-g3 + EGFP-TWF-1 = 197. (C) Lamellipodia protrusion velocities of wild-type, twf1/twf-2 knockout, and knockout cells expressing EGFP-TWF-1. Data represent individual cells with mean and standard deviations shown. Statistical significances in panels B and D were calculated with Mann-Whitney two-tailed test. ****, p
    Figure Legend Snippet: Knockout of twinfilins leads to abnormal F-actin accumulation in lamellipodia and perinuclear region. (A) Representative images of wild-type and twf1/twf2-KO mouse B16-F1 cells stained with AlexaFluor-568 phalloidin and anti-p34 antibody to visualize F-actin and the Arp2/3 complex, respectively. Scale bar = 10 μ m. (B) F-actin intensities in the cytoplasmic regions of wild-type, twf1/twf2-KO, and knockout cells expressing EGFP-TWF-1 measured by high-content image analysis. Number of cells analyzed were: B16-F1 wt = 4,875, twf1/twf2-KO-g3 = 5,731, twf1/twf2-KO-g3 + EGFP-TWF-1 = 197. (C) Lamellipodia protrusion velocities of wild-type, twf1/twf-2 knockout, and knockout cells expressing EGFP-TWF-1. Data represent individual cells with mean and standard deviations shown. Statistical significances in panels B and D were calculated with Mann-Whitney two-tailed test. ****, p

    Techniques Used: Knock-Out, Staining, Expressing, MANN-WHITNEY, Two Tailed Test

    23) Product Images from "Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks"

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    Journal: bioRxiv

    doi: 10.1101/864769

    Twinfilin-1/twinfilin-2 knockout leads to an accumulation of F-actin on endosomes at the perinuclear region. (A) Phalloidin staining of wild-type B16-F1, and (B) twf1/twf2-KO cells after 7.5 min uptake of 20 μ g/ml AlexaFluor-647 transferrin. Scale bar = 20 μ m. The dotted square indicates the perinuclear region magnified in the insert. (C) Mean F-actin intensities in transferrin-positive endosomes of wild-type and twf1/twf2-KO cells as measured from AlexaFluor-555 phalloidin stained cells after 7.5 min intake of 20 μ g/ml AlexaFluor-647 phalloidin. Numbers of measured cells were: B16-F1 wt = 2,518, twf1/2-KO-g3 = 3,637, twf1/2-KO-g3 + EGFP-TWF1 = 158. Statistical significances were calculated with Mann-Whitney two-tailed test. ****, p
    Figure Legend Snippet: Twinfilin-1/twinfilin-2 knockout leads to an accumulation of F-actin on endosomes at the perinuclear region. (A) Phalloidin staining of wild-type B16-F1, and (B) twf1/twf2-KO cells after 7.5 min uptake of 20 μ g/ml AlexaFluor-647 transferrin. Scale bar = 20 μ m. The dotted square indicates the perinuclear region magnified in the insert. (C) Mean F-actin intensities in transferrin-positive endosomes of wild-type and twf1/twf2-KO cells as measured from AlexaFluor-555 phalloidin stained cells after 7.5 min intake of 20 μ g/ml AlexaFluor-647 phalloidin. Numbers of measured cells were: B16-F1 wt = 2,518, twf1/2-KO-g3 = 3,637, twf1/2-KO-g3 + EGFP-TWF1 = 158. Statistical significances were calculated with Mann-Whitney two-tailed test. ****, p

    Techniques Used: Knock-Out, Staining, MANN-WHITNEY, Two Tailed Test

    24) Product Images from "Cellular prion protein is present in mitochondria of healthy mice"

    Article Title: Cellular prion protein is present in mitochondria of healthy mice

    Journal: Scientific Reports

    doi: 10.1038/srep41556

    High resolution confocal microscopy of PrP C co-localization with the mitochondrial protein COXIV. High-resolution confocal images were acquired from formalin fixed and paraffin embedded sagittal sections of cortex co-stained for COXIV and PrP C as detailed in the Methods. Left-hand panels ( a and b ) show X-Y projections of confocal sections (0.16 mm each) from the subset of z-slices indicated in the lower left corner of each panel. Close-up images from a single z-slice of the boxed regions are shown on the right-hand. Yellow arrowheads indicate areas of co-localization. Lower panels ( c ) show no primary controls (Red = streptavidin conjugated AlexaFluor 568; Green = AlexaFluor 488) for the indicated z-slices and were acquired using the same settings as in panels ( a ) and ( b ). Scale bars are shown on the lower right.
    Figure Legend Snippet: High resolution confocal microscopy of PrP C co-localization with the mitochondrial protein COXIV. High-resolution confocal images were acquired from formalin fixed and paraffin embedded sagittal sections of cortex co-stained for COXIV and PrP C as detailed in the Methods. Left-hand panels ( a and b ) show X-Y projections of confocal sections (0.16 mm each) from the subset of z-slices indicated in the lower left corner of each panel. Close-up images from a single z-slice of the boxed regions are shown on the right-hand. Yellow arrowheads indicate areas of co-localization. Lower panels ( c ) show no primary controls (Red = streptavidin conjugated AlexaFluor 568; Green = AlexaFluor 488) for the indicated z-slices and were acquired using the same settings as in panels ( a ) and ( b ). Scale bars are shown on the lower right.

    Techniques Used: Confocal Microscopy, Staining

    25) Product Images from "Absence of TGFβ signaling in retinal microglia induces retinal degeneration and exacerbates choroidal neovascularization"

    Article Title: Absence of TGFβ signaling in retinal microglia induces retinal degeneration and exacerbates choroidal neovascularization

    Journal: eLife

    doi: 10.7554/eLife.42049

    Specific TGFBR2 ablation in retinal microglia induces rapid and progressive changes in microglial morphology and distribution. The time course of morphological changes in retinal microglia following tamoxifen (TMX)-induced ablation of TGFBR2 expression was followed using immunohistochemical analysis in retinal flat-mounts. Panels show changes at the level of the OPL; microglia were labeled using an antibody to IBA1 and retinal vessels labeled with IB4. Gliotic changes in radial Müller glia processes were marked using an antibody to GFAP. At 1 day following TMX administration, a slight reduction in ramification in microglia processes was observed. From 2–5 days post-TMX, a further decrease in microglial ramification and an increase in microglia numbers were detected. From 3–10 weeks post-TMX, retinal microglia transitioned to a branched morphology, demonstrating a close fasciculation with the retinal vasculature. GFAP immunopositivity in Müller glia was prominently upregulated at this time. Scale bar = 100 µm.
    Figure Legend Snippet: Specific TGFBR2 ablation in retinal microglia induces rapid and progressive changes in microglial morphology and distribution. The time course of morphological changes in retinal microglia following tamoxifen (TMX)-induced ablation of TGFBR2 expression was followed using immunohistochemical analysis in retinal flat-mounts. Panels show changes at the level of the OPL; microglia were labeled using an antibody to IBA1 and retinal vessels labeled with IB4. Gliotic changes in radial Müller glia processes were marked using an antibody to GFAP. At 1 day following TMX administration, a slight reduction in ramification in microglia processes was observed. From 2–5 days post-TMX, a further decrease in microglial ramification and an increase in microglia numbers were detected. From 3–10 weeks post-TMX, retinal microglia transitioned to a branched morphology, demonstrating a close fasciculation with the retinal vasculature. GFAP immunopositivity in Müller glia was prominently upregulated at this time. Scale bar = 100 µm.

    Techniques Used: Expressing, Immunohistochemistry, Labeling

    TGFBR2 ablation in retinal microglia induces Müller cell gliosis in the retina. ( A ) Immunohistochemical analysis demonstrates upregulation of immunopositivity to GFAP 3 weeks post-TMX in TG animals relative to control animals. GFAP immunopositivity was localized to glutamine synthetase (GS)-labeled Müller cell processes, indicating the induction of Müller cell gliosis. Scale bar = 50 µm. ( B ) qPCR analysis of retinas isolated from control and TG animals 2 and 8 weeks post-TMX demonstrates a significant upregulation of GFAP mRNA expression following TGFBR2 ablation in retinal microglia. Graphical data are presented as means ± SEM; p values are from one-way analysis of variance (ANOVA) and Sidak’s multiple comparison test, n = 3 animals of mixed sex in each group.( C ) RT-PCR analysis of retinal expression of genes associated with A1- and A2-specific astrocytic gliosis following microglial TGFBR2 ablation found progressive upregulation of A1-associated transcripts relative to control, while A2-associated transcripts were relatively unchanged (numbers indicate means, *, **, *** indicate p values
    Figure Legend Snippet: TGFBR2 ablation in retinal microglia induces Müller cell gliosis in the retina. ( A ) Immunohistochemical analysis demonstrates upregulation of immunopositivity to GFAP 3 weeks post-TMX in TG animals relative to control animals. GFAP immunopositivity was localized to glutamine synthetase (GS)-labeled Müller cell processes, indicating the induction of Müller cell gliosis. Scale bar = 50 µm. ( B ) qPCR analysis of retinas isolated from control and TG animals 2 and 8 weeks post-TMX demonstrates a significant upregulation of GFAP mRNA expression following TGFBR2 ablation in retinal microglia. Graphical data are presented as means ± SEM; p values are from one-way analysis of variance (ANOVA) and Sidak’s multiple comparison test, n = 3 animals of mixed sex in each group.( C ) RT-PCR analysis of retinal expression of genes associated with A1- and A2-specific astrocytic gliosis following microglial TGFBR2 ablation found progressive upregulation of A1-associated transcripts relative to control, while A2-associated transcripts were relatively unchanged (numbers indicate means, *, **, *** indicate p values

    Techniques Used: Immunohistochemistry, Labeling, Real-time Polymerase Chain Reaction, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction

    26) Product Images from "Non-peptidergic primary afferents are presynaptic to neurokinin-1 receptor immunoreactive lamina I projection neurons in rat spinal cord"

    Article Title: Non-peptidergic primary afferents are presynaptic to neurokinin-1 receptor immunoreactive lamina I projection neurons in rat spinal cord

    Journal: Molecular Pain

    doi: 10.1186/1744-8069-8-64

    Example of a quadruple labeling observed at the confocal level using a multi-track approach. In this image the following signals were simultaneously detected: CGRP (green); IB4 binding (red); CTb transported retrogradely from the parabrachial nucleus (blue); NK-1r (white). A fusiform neuron, double labeled with CTb and NK-1r, is innervated by CGRP-IR boutons (arrowhead) and IB4+ (arrow) boutons, which represent distinct populations. However, a small population of varicosities co-labeled for CGRP and IB4 (curved arrow) was detected. Scale bar ( A-E ) = 20 μm.
    Figure Legend Snippet: Example of a quadruple labeling observed at the confocal level using a multi-track approach. In this image the following signals were simultaneously detected: CGRP (green); IB4 binding (red); CTb transported retrogradely from the parabrachial nucleus (blue); NK-1r (white). A fusiform neuron, double labeled with CTb and NK-1r, is innervated by CGRP-IR boutons (arrowhead) and IB4+ (arrow) boutons, which represent distinct populations. However, a small population of varicosities co-labeled for CGRP and IB4 (curved arrow) was detected. Scale bar ( A-E ) = 20 μm.

    Techniques Used: Labeling, Binding Assay, CtB Assay

    Confocal images at high magnification obtained from parasagittal spinal cord sections showing CGRP-IR (green) and P2X3-IR (red) varicosities in the superficial dorsal horn. P2X3-IR varicosities were present in considerable number in lamina I (LI) but were more highly concentrated in inner lamina II (LIIi). Scale bar = 20 μm.
    Figure Legend Snippet: Confocal images at high magnification obtained from parasagittal spinal cord sections showing CGRP-IR (green) and P2X3-IR (red) varicosities in the superficial dorsal horn. P2X3-IR varicosities were present in considerable number in lamina I (LI) but were more highly concentrated in inner lamina II (LIIi). Scale bar = 20 μm.

    Techniques Used:

    Confocal images at high power obtained from horizontal spinal cord sections. In a confocal optical section from lamina I adjacent to the white matter ( A ), note the relatively abundant P2X3-IR fibers with varicosities (boutons). CGRP-IR fibers and boutons were considerably more abundant in this lamina. In a confocal optical section from inner lamina II ( B ), note the very high density of P2X3-IR fibers and varicosities, higher than that of CGRP-IR fibers in lamina I. Note that most varicosities display either P2X3 or CGRP immunoreactivity, although some co-localization is observed (yellow). Scale bar ( A, B ) = 20 μm.
    Figure Legend Snippet: Confocal images at high power obtained from horizontal spinal cord sections. In a confocal optical section from lamina I adjacent to the white matter ( A ), note the relatively abundant P2X3-IR fibers with varicosities (boutons). CGRP-IR fibers and boutons were considerably more abundant in this lamina. In a confocal optical section from inner lamina II ( B ), note the very high density of P2X3-IR fibers and varicosities, higher than that of CGRP-IR fibers in lamina I. Note that most varicosities display either P2X3 or CGRP immunoreactivity, although some co-localization is observed (yellow). Scale bar ( A, B ) = 20 μm.

    Techniques Used:

    CGRP, IB4 and P2X3 staining in transverse spinal cord sections. A and B show low magnification confocal images of CGRP-IR and IB4 positive (A) or P2X3-IR (B) fibers. C and D represent high magnification confocal images from the middle third of the latero-medial extent of the superficial dorsal horn. In C , note that there is limited co-localization of IB4 and CGRP (in yellow). Arrowheads show axonal varicosities (boutons) from non-peptidergic fibers in lamina I, which do not co-localize CGRP immunoreactivity. The framed regions in A and B indicate the approximate regions from where C and D , respectively, were obtained (the latter originate from other sections). CGRP (in green); IB4 (in red); P2X3 (in red). Scale bar ( A , B ) = 200 μm; scale bar ( C , D ) = 20 μm.
    Figure Legend Snippet: CGRP, IB4 and P2X3 staining in transverse spinal cord sections. A and B show low magnification confocal images of CGRP-IR and IB4 positive (A) or P2X3-IR (B) fibers. C and D represent high magnification confocal images from the middle third of the latero-medial extent of the superficial dorsal horn. In C , note that there is limited co-localization of IB4 and CGRP (in yellow). Arrowheads show axonal varicosities (boutons) from non-peptidergic fibers in lamina I, which do not co-localize CGRP immunoreactivity. The framed regions in A and B indicate the approximate regions from where C and D , respectively, were obtained (the latter originate from other sections). CGRP (in green); IB4 (in red); P2X3 (in red). Scale bar ( A , B ) = 200 μm; scale bar ( C , D ) = 20 μm.

    Techniques Used: Staining

    27) Product Images from "Tamiflu-Resistant but HA-Mediated Cell-to-Cell Transmission through Apical Membranes of Cell-Associated Influenza Viruses"

    Article Title: Tamiflu-Resistant but HA-Mediated Cell-to-Cell Transmission through Apical Membranes of Cell-Associated Influenza Viruses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028178

    Influenza viruses can spread independent of the NA activity. (A) MDCK cells were infected with influenza virus A/WSN/33 at a multiplicity of infection (MOI) of 0.001 PFU per cell. At 48 hours post infection (hpi), culture supernatant was collected, and then its virus titer was determined by plaque assays. Each result was represented by a value relative to that in the absence of the drug. Error bars indicate standard deviation (s.d.) from 3 independent experiments. (B) Confluent MDCK cells were infected by wild-type influenza virus A/WSN/33 or NA-deficient influenza virus at MOI of 0.0001 in the presence or absence of 50 µg/ml oseltamivir phosphate. NA-deficient influenza virus was generated by reverse genetics as previously described [29] . After incubation at 37°C for 36 hours, immunofluorescence analyses were performed using anti-nucleoprotein (NP) polyclonal antibody and anti-rabbit IgG antibody conjugated to Alexa Fluor 568 (Invitrogen). Scale bar, 100 µm.
    Figure Legend Snippet: Influenza viruses can spread independent of the NA activity. (A) MDCK cells were infected with influenza virus A/WSN/33 at a multiplicity of infection (MOI) of 0.001 PFU per cell. At 48 hours post infection (hpi), culture supernatant was collected, and then its virus titer was determined by plaque assays. Each result was represented by a value relative to that in the absence of the drug. Error bars indicate standard deviation (s.d.) from 3 independent experiments. (B) Confluent MDCK cells were infected by wild-type influenza virus A/WSN/33 or NA-deficient influenza virus at MOI of 0.0001 in the presence or absence of 50 µg/ml oseltamivir phosphate. NA-deficient influenza virus was generated by reverse genetics as previously described [29] . After incubation at 37°C for 36 hours, immunofluorescence analyses were performed using anti-nucleoprotein (NP) polyclonal antibody and anti-rabbit IgG antibody conjugated to Alexa Fluor 568 (Invitrogen). Scale bar, 100 µm.

    Techniques Used: Activity Assay, Infection, Standard Deviation, Generated, Incubation, Immunofluorescence

    28) Product Images from "Constitutively Active TRPC3 Channels Regulate Basal Ganglia Output Neurons"

    Article Title: Constitutively Active TRPC3 Channels Regulate Basal Ganglia Output Neurons

    Journal:

    doi: 10.1523/JNEUROSCI.3978-07.2008

    TRPC3 channel protein immunoreactivity in SNr GABA neurons. A , A1 shows numerous TRPC3 immunoreactivity-positive, Alexa Fluor 568 (red)-tagged neurons in a 50 μ m-thick SNr section. The boxed area is enlarged and shown in A2 . The somata are clearly
    Figure Legend Snippet: TRPC3 channel protein immunoreactivity in SNr GABA neurons. A , A1 shows numerous TRPC3 immunoreactivity-positive, Alexa Fluor 568 (red)-tagged neurons in a 50 μ m-thick SNr section. The boxed area is enlarged and shown in A2 . The somata are clearly

    Techniques Used:

    Related Articles

    Immunohistochemistry:

    Article Title: Parasitic insect-derived miRNAs modulate host development
    Article Snippet: .. Immunohistochemistry For Alexa-NHS labeling, teratocytes were collected and cultured with water-soluble Alexa Fluor™ 568 NHS Ester (Thermo Fisher Scientific, Hudson, NH, USA) (1 μM) in TNM-FH medium with 10% FBS medium for 30 min. .. The medium was removed and the cells were washed with PBS three times followed by addition of fresh TNM-FH medium with exosome-depleted FBS and cultured for 6 h. The culture medium was then collected and subjected to exosome isolation using an exosome isolation reagent (Invitrogen).

    Recombinant:

    Article Title: Peptides interfering with protein-protein interactions in the ethylene signaling pathway delay tomato fruit ripening
    Article Snippet: .. Quantitative analysis of inhibitory peptides on ETR1-EIN2 complex by FRET For the FRET assay, recombinant LeETR1 was labeled with Alexa Fluor 488 succinimidyl-ester and LeEIN2462–1316 was labeled with Alexa Fluor 568 succinimidyl-ester (both Life Technologies) in a buffer containing 50 mM potassium phosphate pH 8.0, 300 mM NaCl and 5% (v/v) glycerol following manufacturer’s protocol (MolecularProbes Protein Labeling Kit). .. Labeled proteins were transferred in FRET buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.05% (v/v) Tween 20, 5% (v/v) glycerol and mixed at a final concentration of 3.0 μM each.

    Labeling:

    Article Title: Peptides interfering with protein-protein interactions in the ethylene signaling pathway delay tomato fruit ripening
    Article Snippet: .. Quantitative analysis of inhibitory peptides on ETR1-EIN2 complex by FRET For the FRET assay, recombinant LeETR1 was labeled with Alexa Fluor 488 succinimidyl-ester and LeEIN2462–1316 was labeled with Alexa Fluor 568 succinimidyl-ester (both Life Technologies) in a buffer containing 50 mM potassium phosphate pH 8.0, 300 mM NaCl and 5% (v/v) glycerol following manufacturer’s protocol (MolecularProbes Protein Labeling Kit). .. Labeled proteins were transferred in FRET buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.05% (v/v) Tween 20, 5% (v/v) glycerol and mixed at a final concentration of 3.0 μM each.

    Article Title: The transduction of Coxsackie and Adenovirus Receptor-negative cells and protection against neutralizing antibodies by HPMA-co-oligolysine copolymer-coated adenovirus
    Article Snippet: .. Alexa Fluor 568-labeled Ad5-GFP was prepared by labeling 1 × 1012 vp of Ad5-GFP with five-fold excess of Alexa Fluor 568 succinimidyl ester (Invitrogen, Carlsbad, CA) with respect to the surface lysines present on the capsid (assumed to be 18,000 from ( )). .. The reaction was performed in sodium bicarbonate buffer (final concentration 50 mM, pH 8.0) at room temperature for one hour.

    Article Title: An in vitro assay for entry into cilia reveals unique properties of the soluble diffusion barrier
    Article Snippet: .. Peak fractions were pooled and labeled with Alexa Fluor 647 or Alexa Fluor 568 succinimidyl esters according to the manufacturer’s protocol (Life Technologies), and excess dye was separated from labeled protein with columns (NAP-5; GE Healthcare). .. Semipermeabilization with digitonin and PFO Coverslips with serum-starved IMCD3 cells were first placed on an ice-chilled metal block and washed twice with cold assay buffer (20 mM Hepes, pH 7.4, 115 mM KOAc, 1 mM MgCl2 , and 1 mM EGTA).

    Article Title: Parasitic insect-derived miRNAs modulate host development
    Article Snippet: .. Immunohistochemistry For Alexa-NHS labeling, teratocytes were collected and cultured with water-soluble Alexa Fluor™ 568 NHS Ester (Thermo Fisher Scientific, Hudson, NH, USA) (1 μM) in TNM-FH medium with 10% FBS medium for 30 min. .. The medium was removed and the cells were washed with PBS three times followed by addition of fresh TNM-FH medium with exosome-depleted FBS and cultured for 6 h. The culture medium was then collected and subjected to exosome isolation using an exosome isolation reagent (Invitrogen).

    Article Title: Autophagy promotes degradation of polyethyleneimine–alginate nanoparticles in endothelial progenitor cells
    Article Snippet: .. Labeling of lysosomes The cells were treated with Alexa Fluor 568 N-hydroxysuccinimide (NHS) Ester (Thermo Fisher)-labeled PEI–Alg NPs for 4 h at 37°C. .. After washing with PBS, the cells were incubated with 50 nM LysoTracker Green DND-26 (Thermo Fisher Scientific) for 30 min. Then, the cells were washed with ice-cold PBS for three times.

    Article Title: Parasitic insect-derived miRNAs modulate host development
    Article Snippet: .. For Alexa-NHS labeling, teratocytes were collected and cultured with water-soluble Alexa Fluor™ 568 NHS Ester (Thermo Fisher Scientific, Hudson, NH, USA) (1 μM) in TNM-FH medium with 10% FBS medium for 30 min. .. The medium was removed and the cells were washed with PBS three times followed by addition of fresh TNM-FH medium with exosome-depleted FBS and cultured for 6 h. The culture medium was then collected and subjected to exosome isolation using an exosome isolation reagent (Invitrogen).

    Cell Culture:

    Article Title: Parasitic insect-derived miRNAs modulate host development
    Article Snippet: .. Immunohistochemistry For Alexa-NHS labeling, teratocytes were collected and cultured with water-soluble Alexa Fluor™ 568 NHS Ester (Thermo Fisher Scientific, Hudson, NH, USA) (1 μM) in TNM-FH medium with 10% FBS medium for 30 min. .. The medium was removed and the cells were washed with PBS three times followed by addition of fresh TNM-FH medium with exosome-depleted FBS and cultured for 6 h. The culture medium was then collected and subjected to exosome isolation using an exosome isolation reagent (Invitrogen).

    Article Title: Parasitic insect-derived miRNAs modulate host development
    Article Snippet: .. For Alexa-NHS labeling, teratocytes were collected and cultured with water-soluble Alexa Fluor™ 568 NHS Ester (Thermo Fisher Scientific, Hudson, NH, USA) (1 μM) in TNM-FH medium with 10% FBS medium for 30 min. .. The medium was removed and the cells were washed with PBS three times followed by addition of fresh TNM-FH medium with exosome-depleted FBS and cultured for 6 h. The culture medium was then collected and subjected to exosome isolation using an exosome isolation reagent (Invitrogen).

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    Thermo Fisher goat anti rabbit igg alexafluor 568 conjugated secondary antibody
    Twinfilin regulates capping protein localization and dynamics. (A) Localization of EGFP-CP in wild-type and twf1/twf2-KO B16-F1 cells, where F-actin was visualized with <t>AlexaFluor-568</t> phalloidin. Panels in the middle and right are magnifications of lamellipodial regions highlighted in the whole cell images in left. Scale bars = 10 μ m. (B) Examples of line profiles generated across the center of lamellipodia as indicated with dotted lines. Data represent mean of 5 measurements of individual lamellipodia, with standard deviations shown. The ‘0 μ m’ value in x-axis is set to correspond the peak intensity of phalloidin. (C) The ratio of CP and F-actin co-localization widths were detected by measuring the width of localization at 50% of maximum intensity. Data points represent measurements from individual lamellipodia with mean values and standard deviations shown. Statistical significance was calculated with Student’s unpaired, two-tailed t-test. ****, p
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    Twinfilin regulates capping protein localization and dynamics. (A) Localization of EGFP-CP in wild-type and twf1/twf2-KO B16-F1 cells, where F-actin was visualized with AlexaFluor-568 phalloidin. Panels in the middle and right are magnifications of lamellipodial regions highlighted in the whole cell images in left. Scale bars = 10 μ m. (B) Examples of line profiles generated across the center of lamellipodia as indicated with dotted lines. Data represent mean of 5 measurements of individual lamellipodia, with standard deviations shown. The ‘0 μ m’ value in x-axis is set to correspond the peak intensity of phalloidin. (C) The ratio of CP and F-actin co-localization widths were detected by measuring the width of localization at 50% of maximum intensity. Data points represent measurements from individual lamellipodia with mean values and standard deviations shown. Statistical significance was calculated with Student’s unpaired, two-tailed t-test. ****, p

    Journal: bioRxiv

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    doi: 10.1101/864769

    Figure Lengend Snippet: Twinfilin regulates capping protein localization and dynamics. (A) Localization of EGFP-CP in wild-type and twf1/twf2-KO B16-F1 cells, where F-actin was visualized with AlexaFluor-568 phalloidin. Panels in the middle and right are magnifications of lamellipodial regions highlighted in the whole cell images in left. Scale bars = 10 μ m. (B) Examples of line profiles generated across the center of lamellipodia as indicated with dotted lines. Data represent mean of 5 measurements of individual lamellipodia, with standard deviations shown. The ‘0 μ m’ value in x-axis is set to correspond the peak intensity of phalloidin. (C) The ratio of CP and F-actin co-localization widths were detected by measuring the width of localization at 50% of maximum intensity. Data points represent measurements from individual lamellipodia with mean values and standard deviations shown. Statistical significance was calculated with Student’s unpaired, two-tailed t-test. ****, p

    Article Snippet: Other antibodies used in the study were: Rabbit anti-twinfilin-2 antibody (Sigma-Aldrich #HPA053874, WB, 1:100), rabbit anti-CAPZß antibody (Sigma-Aldrich, #HPA031531, WB, 1:100), mouse anti-α-tubulin antibody (Sigma-Aldrich, #T5168, WB 1:10,000), mouse anti-ß-actin antibody (Sigma-Aldrich, #A5441, WB, 1:10,000), Rabbit anti-p34-Arc/ARPC2 (Merck Millipore, #07-227, dilution in immunofluorescence (IF), 1:200), goat anti-Rabbit IgG AlexaFluor-488 conjugated secondary antibody (Thermo Fisher, #A-11034, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-568 conjugated secondary antibody (Thermo Fisher, #A-11011, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-647 conjugated secondary antibody (Thermo Fisher, #A-32733, IF, 1:400), goat anti-Mouse IgG HRP conjugated secondary antibody (Thermo Fisher, #31430, WB, 1:10,000), goat anti-Rabbit IgG HRP conjugated secondary antibody (Thermo Fisher, #32460, WB, 1:1,000).

    Techniques: Generated, Two Tailed Test

    Examples of high-content and lamellipodia protrusion analysis. (A) Representative images of segmentation procedure in high-content image analysis. Wild-type and twinfilin-deficient B16-F1 cells were stained with DAPI and CellMask Deep Red to segment nuclei (outlined with blue) and cytoplasm (outlined with yellow). The Arp2/3-complex positive structures were detected with anti-p34 antibody staining and used as a mask for the Arp2/3-complex positive F-actin structures (the most-right panel). F-actin was stained with AlexaFluor-568 phalloidin. Cells touching the border of images were excluded from analysis. (B) A representative example of twf1/twf2-KO cell migrating on laminin coated glass imaged with DIC. Direction of migration is indicated with an arrow and kymographs were generated with line drawn across the lamellipodium as indicated with dotted red line. (C) Representative examples of kymographs generated from DIC time-lapse images of wild-type, twf1/twf2 knockout, and EGFP-TWF-1 rescue cells. Protrusions velocities were measured from the overall cell front protrusion as indicated with dotted red lines.

    Journal: bioRxiv

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    doi: 10.1101/864769

    Figure Lengend Snippet: Examples of high-content and lamellipodia protrusion analysis. (A) Representative images of segmentation procedure in high-content image analysis. Wild-type and twinfilin-deficient B16-F1 cells were stained with DAPI and CellMask Deep Red to segment nuclei (outlined with blue) and cytoplasm (outlined with yellow). The Arp2/3-complex positive structures were detected with anti-p34 antibody staining and used as a mask for the Arp2/3-complex positive F-actin structures (the most-right panel). F-actin was stained with AlexaFluor-568 phalloidin. Cells touching the border of images were excluded from analysis. (B) A representative example of twf1/twf2-KO cell migrating on laminin coated glass imaged with DIC. Direction of migration is indicated with an arrow and kymographs were generated with line drawn across the lamellipodium as indicated with dotted red line. (C) Representative examples of kymographs generated from DIC time-lapse images of wild-type, twf1/twf2 knockout, and EGFP-TWF-1 rescue cells. Protrusions velocities were measured from the overall cell front protrusion as indicated with dotted red lines.

    Article Snippet: Other antibodies used in the study were: Rabbit anti-twinfilin-2 antibody (Sigma-Aldrich #HPA053874, WB, 1:100), rabbit anti-CAPZß antibody (Sigma-Aldrich, #HPA031531, WB, 1:100), mouse anti-α-tubulin antibody (Sigma-Aldrich, #T5168, WB 1:10,000), mouse anti-ß-actin antibody (Sigma-Aldrich, #A5441, WB, 1:10,000), Rabbit anti-p34-Arc/ARPC2 (Merck Millipore, #07-227, dilution in immunofluorescence (IF), 1:200), goat anti-Rabbit IgG AlexaFluor-488 conjugated secondary antibody (Thermo Fisher, #A-11034, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-568 conjugated secondary antibody (Thermo Fisher, #A-11011, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-647 conjugated secondary antibody (Thermo Fisher, #A-32733, IF, 1:400), goat anti-Mouse IgG HRP conjugated secondary antibody (Thermo Fisher, #31430, WB, 1:10,000), goat anti-Rabbit IgG HRP conjugated secondary antibody (Thermo Fisher, #32460, WB, 1:1,000).

    Techniques: Staining, Migration, Generated, Knock-Out

    Representative images of wild-type and twinfilin knockout B16-F1 cells. (A) B16-F1 cells were stained with AlexaFluor-568 phalloidin (F-actin) and anti-p34 antibody (the Arp2/3 complex). Scale bars = 10 μ M. (B) Mean F-actin intensity in B16-F1 wild-type and twinfilin knockout cells. Number of measured cells were: B16-F1 wt = 1,958, twf1-KO-g1 = 1,045, twf2-KO-g3#1 = 1,285, twf1/2-KO-g3 = 1,265, twf1/2-KO-g4 = 1,707. (C) Mean F-actin intensity in the Arp2/3 complex positive regions of B16-F1 wild-type and knockout cells. The Arp2/3 complex positive regions were identified based on p34-antibody staining. Number of measured cells were: B16-F1 wt = 1,658, twf1-KO-g1 = 884, twf2-KO-g3#1 = 1,009, twf1/2-KO-g3 = 1,100, twf1/2-KO-g4 = 1157. Statistical significances in panels B and C were calculated with Mann-Whitney two-tailed test. ****, p

    Journal: bioRxiv

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    doi: 10.1101/864769

    Figure Lengend Snippet: Representative images of wild-type and twinfilin knockout B16-F1 cells. (A) B16-F1 cells were stained with AlexaFluor-568 phalloidin (F-actin) and anti-p34 antibody (the Arp2/3 complex). Scale bars = 10 μ M. (B) Mean F-actin intensity in B16-F1 wild-type and twinfilin knockout cells. Number of measured cells were: B16-F1 wt = 1,958, twf1-KO-g1 = 1,045, twf2-KO-g3#1 = 1,285, twf1/2-KO-g3 = 1,265, twf1/2-KO-g4 = 1,707. (C) Mean F-actin intensity in the Arp2/3 complex positive regions of B16-F1 wild-type and knockout cells. The Arp2/3 complex positive regions were identified based on p34-antibody staining. Number of measured cells were: B16-F1 wt = 1,658, twf1-KO-g1 = 884, twf2-KO-g3#1 = 1,009, twf1/2-KO-g3 = 1,100, twf1/2-KO-g4 = 1157. Statistical significances in panels B and C were calculated with Mann-Whitney two-tailed test. ****, p

    Article Snippet: Other antibodies used in the study were: Rabbit anti-twinfilin-2 antibody (Sigma-Aldrich #HPA053874, WB, 1:100), rabbit anti-CAPZß antibody (Sigma-Aldrich, #HPA031531, WB, 1:100), mouse anti-α-tubulin antibody (Sigma-Aldrich, #T5168, WB 1:10,000), mouse anti-ß-actin antibody (Sigma-Aldrich, #A5441, WB, 1:10,000), Rabbit anti-p34-Arc/ARPC2 (Merck Millipore, #07-227, dilution in immunofluorescence (IF), 1:200), goat anti-Rabbit IgG AlexaFluor-488 conjugated secondary antibody (Thermo Fisher, #A-11034, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-568 conjugated secondary antibody (Thermo Fisher, #A-11011, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-647 conjugated secondary antibody (Thermo Fisher, #A-32733, IF, 1:400), goat anti-Mouse IgG HRP conjugated secondary antibody (Thermo Fisher, #31430, WB, 1:10,000), goat anti-Rabbit IgG HRP conjugated secondary antibody (Thermo Fisher, #32460, WB, 1:1,000).

    Techniques: Knock-Out, Staining, MANN-WHITNEY, Two Tailed Test

    Knockout of twinfilins leads to abnormal F-actin accumulation in lamellipodia and perinuclear region. (A) Representative images of wild-type and twf1/twf2-KO mouse B16-F1 cells stained with AlexaFluor-568 phalloidin and anti-p34 antibody to visualize F-actin and the Arp2/3 complex, respectively. Scale bar = 10 μ m. (B) F-actin intensities in the cytoplasmic regions of wild-type, twf1/twf2-KO, and knockout cells expressing EGFP-TWF-1 measured by high-content image analysis. Number of cells analyzed were: B16-F1 wt = 4,875, twf1/twf2-KO-g3 = 5,731, twf1/twf2-KO-g3 + EGFP-TWF-1 = 197. (C) Lamellipodia protrusion velocities of wild-type, twf1/twf-2 knockout, and knockout cells expressing EGFP-TWF-1. Data represent individual cells with mean and standard deviations shown. Statistical significances in panels B and D were calculated with Mann-Whitney two-tailed test. ****, p

    Journal: bioRxiv

    Article Title: Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

    doi: 10.1101/864769

    Figure Lengend Snippet: Knockout of twinfilins leads to abnormal F-actin accumulation in lamellipodia and perinuclear region. (A) Representative images of wild-type and twf1/twf2-KO mouse B16-F1 cells stained with AlexaFluor-568 phalloidin and anti-p34 antibody to visualize F-actin and the Arp2/3 complex, respectively. Scale bar = 10 μ m. (B) F-actin intensities in the cytoplasmic regions of wild-type, twf1/twf2-KO, and knockout cells expressing EGFP-TWF-1 measured by high-content image analysis. Number of cells analyzed were: B16-F1 wt = 4,875, twf1/twf2-KO-g3 = 5,731, twf1/twf2-KO-g3 + EGFP-TWF-1 = 197. (C) Lamellipodia protrusion velocities of wild-type, twf1/twf-2 knockout, and knockout cells expressing EGFP-TWF-1. Data represent individual cells with mean and standard deviations shown. Statistical significances in panels B and D were calculated with Mann-Whitney two-tailed test. ****, p

    Article Snippet: Other antibodies used in the study were: Rabbit anti-twinfilin-2 antibody (Sigma-Aldrich #HPA053874, WB, 1:100), rabbit anti-CAPZß antibody (Sigma-Aldrich, #HPA031531, WB, 1:100), mouse anti-α-tubulin antibody (Sigma-Aldrich, #T5168, WB 1:10,000), mouse anti-ß-actin antibody (Sigma-Aldrich, #A5441, WB, 1:10,000), Rabbit anti-p34-Arc/ARPC2 (Merck Millipore, #07-227, dilution in immunofluorescence (IF), 1:200), goat anti-Rabbit IgG AlexaFluor-488 conjugated secondary antibody (Thermo Fisher, #A-11034, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-568 conjugated secondary antibody (Thermo Fisher, #A-11011, IF, 1:400), goat anti-Rabbit IgG AlexaFluor-647 conjugated secondary antibody (Thermo Fisher, #A-32733, IF, 1:400), goat anti-Mouse IgG HRP conjugated secondary antibody (Thermo Fisher, #31430, WB, 1:10,000), goat anti-Rabbit IgG HRP conjugated secondary antibody (Thermo Fisher, #32460, WB, 1:1,000).

    Techniques: Knock-Out, Staining, Expressing, MANN-WHITNEY, Two Tailed Test