dapi  (Vector Laboratories)


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    Name:
    VECTASHIELD Antifade Mounting Medium
    Description:
    VECTASHIELD Antifade Mounting Medium is a unique stable formula for preserving fluorescence VECTASHIELD Mounting Medium prevents rapid photobleaching of fluorescent proteins and fluorescent dyes Features Inhibits photobleaching of dyes and fluorescent proteins Ideal refractive index Ready to use Offered with nuclear or cytoskeletal counterstains Available in non hardening or hardening formulations No warming necessary Can be stored without sealing for long term analysis Easy to useThe original VECTASHIELD Mounting Medium does not solidify but remains a liquid on the slide and can be stored without sealing If desired coverslips can be sealed around the perimeter with nail polish or a plastic sealant Mounted slides should be stored at 4 °C protected from light
    Catalog Number:
    H-1000
    Price:
    None
    Category:
    Histology reagents or solutions or stains
    Size:
    10 ml
    Buy from Supplier


    Structured Review

    Vector Laboratories dapi
    VECTASHIELD Antifade Mounting Medium
    VECTASHIELD Antifade Mounting Medium is a unique stable formula for preserving fluorescence VECTASHIELD Mounting Medium prevents rapid photobleaching of fluorescent proteins and fluorescent dyes Features Inhibits photobleaching of dyes and fluorescent proteins Ideal refractive index Ready to use Offered with nuclear or cytoskeletal counterstains Available in non hardening or hardening formulations No warming necessary Can be stored without sealing for long term analysis Easy to useThe original VECTASHIELD Mounting Medium does not solidify but remains a liquid on the slide and can be stored without sealing If desired coverslips can be sealed around the perimeter with nail polish or a plastic sealant Mounted slides should be stored at 4 °C protected from light
    https://www.bioz.com/result/dapi/product/Vector Laboratories
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dapi - by Bioz Stars, 2021-05
    99/100 stars

    Images

    1) Product Images from "Fgf10-Hippo epithelial mesenchymal crosstalk maintains and recruits lung basal stem cells"

    Article Title: Fgf10-Hippo epithelial mesenchymal crosstalk maintains and recruits lung basal stem cells

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2017.09.003

    Fgf10-Fgfr2b signaling downstream of Yap drives secretory and basal cell amplification in the lower non-cartilaginous conducting airways upon Ilk inactivation (A) Schematic representation of non-cartilaginous airway secretory/ciliated amplification and BSC amplification or loss, stromal Fgf10 or nuclear Yap levels upon airway epithelial Ilk/Fgfr2b ablation, Ilk ablation and Ilk/Yap ablation. All lungs were collected at 2 months after tamoxifen induction. (B) Sirius red (collagen)/fast green staining and immunostaining on Sox2-Ilk f/f -Fgfr2b f/f , Sox2-Ilk f/f , and Sox2-Ilk f/f -Yap f/f non-cartilaginous airways for BSC markers p63 (red) and Keratin 5 (K5) (green). (C,D) Morphometric analysis of airway remodeling (C) and quantification of BSC numbers (#) per 100 μm basement membrane (D) from images represented in (B). (E) Relative mRNA levels of Scgb1a1 (secretory cells), FoxJ1 (ciliated cells), K5 (BSCs), p63 (BSCs) , Col1a1 (collagen) and Col3a1 (collagen) in control, Sox2-Ilk f/f (ILK KO), Sox2-Ilk f/f -Fgfr2b f/f (ILK FGFR2b DKO) and Sox2-Ilk f/f -Yap f/f (ILK YAP DKO) lungs. Nuclei were stained with DAPI (blue). ** P
    Figure Legend Snippet: Fgf10-Fgfr2b signaling downstream of Yap drives secretory and basal cell amplification in the lower non-cartilaginous conducting airways upon Ilk inactivation (A) Schematic representation of non-cartilaginous airway secretory/ciliated amplification and BSC amplification or loss, stromal Fgf10 or nuclear Yap levels upon airway epithelial Ilk/Fgfr2b ablation, Ilk ablation and Ilk/Yap ablation. All lungs were collected at 2 months after tamoxifen induction. (B) Sirius red (collagen)/fast green staining and immunostaining on Sox2-Ilk f/f -Fgfr2b f/f , Sox2-Ilk f/f , and Sox2-Ilk f/f -Yap f/f non-cartilaginous airways for BSC markers p63 (red) and Keratin 5 (K5) (green). (C,D) Morphometric analysis of airway remodeling (C) and quantification of BSC numbers (#) per 100 μm basement membrane (D) from images represented in (B). (E) Relative mRNA levels of Scgb1a1 (secretory cells), FoxJ1 (ciliated cells), K5 (BSCs), p63 (BSCs) , Col1a1 (collagen) and Col3a1 (collagen) in control, Sox2-Ilk f/f (ILK KO), Sox2-Ilk f/f -Fgfr2b f/f (ILK FGFR2b DKO) and Sox2-Ilk f/f -Yap f/f (ILK YAP DKO) lungs. Nuclei were stained with DAPI (blue). ** P

    Techniques Used: Amplification, Staining, Immunostaining

    Inactivation of the Hippo pathway in differentiated airway epithelial cells after injury or after Ilk inactivation induces epithelial Wnt7b expression and Fgf10 secretion by ASMCs (A) Immunostaining on non-cartilaginous airways of non-injured (NI) control lungs and lungs 3 days after naphthalene (npt)-induced injury for secretory cell marker Scgb1a1 (green) or ciliated cell marker β-tubulin (green) and Yap or Merlin (red). (B) Quantification of Yap pixel intensity of pictures represented in (A) ( n ≥ 6 mice). (C) Quantification of Merlin pixel intensity of pictures represented in (A) ( n ≥ 6 mice). (D) Immunostaining on non-cartilaginous airways of control Fgf10 LacZ , Scgb1a1-Mst1/2 f/f -Fgf10 LacZ and Scgb1a1-Ilk f/f -Fgf10 LacZ lungs for secretory cell marker Scgb1a1 (green) with either Yap (red) or Wnt7b (red) or Dkk1 (red) or Fgfr2b (red) and β- gal staining of Fgf10 LacZ . Black arrowheads indicate Fgf10 expression in ASMCs. (E) Quantification of pixel intensity of Yap, Wnt7b, Dkk1 and Fgfr2b signals represented in (D) ( n ≥ 6 mice). (F) Relative mRNA expression of Wnt7b and Dkk1 in control, Scgb1a1-Mst1/2 f/f and Scgb1a1-Ilk f/f lungs. (G) Western blot analysis showing FGF10, YAP, FGFR2B, P-MST1/2, and WNT7B protein expression in control, Scgb1a1-Mst1/2 f/f (MST1/2 KO) and Scgb1a1-Ilk f/f (ILK KO) lungs. (H) Quantification of pixel intensities of pictures represented in (I) ( n ≥ 6 mice). (I) Immunostaining on control and Scgb1a1-Ilk f/f non-cartilaginous airways for Merlin (red) or phospho-Mst1/2 (red). Nuclei, DAPI (blue). ** P
    Figure Legend Snippet: Inactivation of the Hippo pathway in differentiated airway epithelial cells after injury or after Ilk inactivation induces epithelial Wnt7b expression and Fgf10 secretion by ASMCs (A) Immunostaining on non-cartilaginous airways of non-injured (NI) control lungs and lungs 3 days after naphthalene (npt)-induced injury for secretory cell marker Scgb1a1 (green) or ciliated cell marker β-tubulin (green) and Yap or Merlin (red). (B) Quantification of Yap pixel intensity of pictures represented in (A) ( n ≥ 6 mice). (C) Quantification of Merlin pixel intensity of pictures represented in (A) ( n ≥ 6 mice). (D) Immunostaining on non-cartilaginous airways of control Fgf10 LacZ , Scgb1a1-Mst1/2 f/f -Fgf10 LacZ and Scgb1a1-Ilk f/f -Fgf10 LacZ lungs for secretory cell marker Scgb1a1 (green) with either Yap (red) or Wnt7b (red) or Dkk1 (red) or Fgfr2b (red) and β- gal staining of Fgf10 LacZ . Black arrowheads indicate Fgf10 expression in ASMCs. (E) Quantification of pixel intensity of Yap, Wnt7b, Dkk1 and Fgfr2b signals represented in (D) ( n ≥ 6 mice). (F) Relative mRNA expression of Wnt7b and Dkk1 in control, Scgb1a1-Mst1/2 f/f and Scgb1a1-Ilk f/f lungs. (G) Western blot analysis showing FGF10, YAP, FGFR2B, P-MST1/2, and WNT7B protein expression in control, Scgb1a1-Mst1/2 f/f (MST1/2 KO) and Scgb1a1-Ilk f/f (ILK KO) lungs. (H) Quantification of pixel intensities of pictures represented in (I) ( n ≥ 6 mice). (I) Immunostaining on control and Scgb1a1-Ilk f/f non-cartilaginous airways for Merlin (red) or phospho-Mst1/2 (red). Nuclei, DAPI (blue). ** P

    Techniques Used: Expressing, Immunostaining, Marker, Mouse Assay, Staining, Western Blot

    Fgf10 expression in ASMCs drives secretory and basal cell amplification in the lower non-cartilaginous conducting airways upon Ilk inactivation (A) Schematic representation of airway BSC amplification or loss, stromal Fgf10 or nuclear Yap levels in the different mutant strains. (B) Sirius red (collagen)/fast green staining and immunostaining on Scgb1a1-Ilk f/f (ILK KO), Scgb1a1-Ilk f/f -Fgfr2b f/f (ILK FGFR2b DKO) and Scgb1a1-Ilk f/f -rtTa-Dkk1 (ILK DKK1) non-cartilaginous airways for BSC markers p63 (red) and Keratin 5 (K5) (green) or secretory cell marker Scgb1a1 (green) and Yap (red). (C,D) Morphometric analysis of airway remodeling (C) and quantification of BSC numbers (D) of images represented in (B). (E) Relative mRNA levels of Scgb1a1 (secretory cells), FoxJ1 (ciliated cells), K5 (BSCs), p63 (BSCs) , Col1a1 (collagen) and Col3a1 (collagen) in control, Scgb1a1-Ilk f/f , Scgb1a1-Ilk f/f -Fgfr2b f/f and Scgb1a1-Ilk f/f -rtTa-Dkk1 lungs. Nuclei were stained with DAPI (blue). ** P
    Figure Legend Snippet: Fgf10 expression in ASMCs drives secretory and basal cell amplification in the lower non-cartilaginous conducting airways upon Ilk inactivation (A) Schematic representation of airway BSC amplification or loss, stromal Fgf10 or nuclear Yap levels in the different mutant strains. (B) Sirius red (collagen)/fast green staining and immunostaining on Scgb1a1-Ilk f/f (ILK KO), Scgb1a1-Ilk f/f -Fgfr2b f/f (ILK FGFR2b DKO) and Scgb1a1-Ilk f/f -rtTa-Dkk1 (ILK DKK1) non-cartilaginous airways for BSC markers p63 (red) and Keratin 5 (K5) (green) or secretory cell marker Scgb1a1 (green) and Yap (red). (C,D) Morphometric analysis of airway remodeling (C) and quantification of BSC numbers (D) of images represented in (B). (E) Relative mRNA levels of Scgb1a1 (secretory cells), FoxJ1 (ciliated cells), K5 (BSCs), p63 (BSCs) , Col1a1 (collagen) and Col3a1 (collagen) in control, Scgb1a1-Ilk f/f , Scgb1a1-Ilk f/f -Fgfr2b f/f and Scgb1a1-Ilk f/f -rtTa-Dkk1 lungs. Nuclei were stained with DAPI (blue). ** P

    Techniques Used: Expressing, Amplification, Mutagenesis, Staining, Immunostaining, Marker

    The inactive Hippo pathway in basal stem/progenitor cells generates the Fgf10 -expressing tracheal stromal niche required to maintain their cell pool (A) Experimental strategy and schematic representation of tracheal BSC amplification after Fgf10 overexpression or tracheal BSC loss after Fgfr2b ablation in airway epithelial cells. Ciliated, secretory and BSCs are shown in green, blue and red, respectively. (B) Immunostaining on rtTa-Fgf10, control and Sox2-Fgfr2b f/f tracheas for the BSC markers Keratin 5 (K5) (green) and p63 (red) 14 days after doxycycline or tamoxifen induction. (C) Quantification of the number (#) of BSCs per 100 μm basement membrane of pictures represented in (B). (D) Experimental strategy and schematic representation of Mst1/2 ablation in all airway epithelial cells or selectively in secretory/ciliated cells alone or in combination with either Fgfr2b or Yap in all airway epithelial cells with or without simultaneously inducing Fgf10 expression. (E) Whole mount in situ hybridization for Fgf10 in control, Scgb1a1-Mst1/2 f/f and Sox2-Mst1/2 f/f tracheas. Note purple Fgf10 expression between the tracheal cartilage rings. (F) Upper panels show immunostaining on control, Sox2-Mst1/2 f/f and Sox2-Mst1/2 f/f -Fgfr2b f/f tracheas 2 months after tamoxifen induction as well as on a 2.5 month old Scgb1a1-Mst1/2 f/f -Confetti trachea for the BSC marker K5 (red) and the secretory cell marker Scgb1a1 (green). Lower panel shows immunostaining on Scgb1a1-Mst1/2 f/f -Confetti trachea for the BSC marker K5 (red) and GFP (green). (G) Quantification of the number (#) of BSCs per 100 μm basement membrane (BM) of pictures represented in (F,H). (H) Immunostaining on control, Sox2-Yap f/f , Sox2-Yap f/f -rtTa-Fgf10 tracheas for the BSC markers K5 (green) and p63 (red) after tamoxifen-induced deletion of Yap and/or doxycycline-induced Fgf10 expression. Refer to panel D for experimental strategy. (I) Immunostaining on adjacent tracheal sections from control, Sox2-Yap f/f and Sox2-Yap f/f -Fgf10 mice for BSC marker K5 (green) and Yap (red) or proliferation marker PCNA (red) 12 weeks after tamoxifen and 2 weeks after doxycycline induction, starting at 10 weeks after tamoxifen induction. Nuclei, DAPI (blue). ** P
    Figure Legend Snippet: The inactive Hippo pathway in basal stem/progenitor cells generates the Fgf10 -expressing tracheal stromal niche required to maintain their cell pool (A) Experimental strategy and schematic representation of tracheal BSC amplification after Fgf10 overexpression or tracheal BSC loss after Fgfr2b ablation in airway epithelial cells. Ciliated, secretory and BSCs are shown in green, blue and red, respectively. (B) Immunostaining on rtTa-Fgf10, control and Sox2-Fgfr2b f/f tracheas for the BSC markers Keratin 5 (K5) (green) and p63 (red) 14 days after doxycycline or tamoxifen induction. (C) Quantification of the number (#) of BSCs per 100 μm basement membrane of pictures represented in (B). (D) Experimental strategy and schematic representation of Mst1/2 ablation in all airway epithelial cells or selectively in secretory/ciliated cells alone or in combination with either Fgfr2b or Yap in all airway epithelial cells with or without simultaneously inducing Fgf10 expression. (E) Whole mount in situ hybridization for Fgf10 in control, Scgb1a1-Mst1/2 f/f and Sox2-Mst1/2 f/f tracheas. Note purple Fgf10 expression between the tracheal cartilage rings. (F) Upper panels show immunostaining on control, Sox2-Mst1/2 f/f and Sox2-Mst1/2 f/f -Fgfr2b f/f tracheas 2 months after tamoxifen induction as well as on a 2.5 month old Scgb1a1-Mst1/2 f/f -Confetti trachea for the BSC marker K5 (red) and the secretory cell marker Scgb1a1 (green). Lower panel shows immunostaining on Scgb1a1-Mst1/2 f/f -Confetti trachea for the BSC marker K5 (red) and GFP (green). (G) Quantification of the number (#) of BSCs per 100 μm basement membrane (BM) of pictures represented in (F,H). (H) Immunostaining on control, Sox2-Yap f/f , Sox2-Yap f/f -rtTa-Fgf10 tracheas for the BSC markers K5 (green) and p63 (red) after tamoxifen-induced deletion of Yap and/or doxycycline-induced Fgf10 expression. Refer to panel D for experimental strategy. (I) Immunostaining on adjacent tracheal sections from control, Sox2-Yap f/f and Sox2-Yap f/f -Fgf10 mice for BSC marker K5 (green) and Yap (red) or proliferation marker PCNA (red) 12 weeks after tamoxifen and 2 weeks after doxycycline induction, starting at 10 weeks after tamoxifen induction. Nuclei, DAPI (blue). ** P

    Techniques Used: Expressing, Amplification, Over Expression, Immunostaining, In Situ Hybridization, Marker, Mouse Assay

    2) Product Images from "Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations"

    Article Title: Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations

    Journal: Scientific Reports

    doi: 10.1038/srep16279

    Correlating 3D-SIM and d STORM images of a rat liver sinusoidal endothelial cell (LSEC). ( A ) Maximum intensity z-projection 3D-SIM image of a 4-color-stained fixed rat LSEC. The nucleus was stained with DAPI (blue), actin filaments with Phalloidin-Alexa488 (green), membranes with CellMask Orange (white), and tubulin structures with anti β-tubulin mouse antibody followed by an anti-mouse IgG-Alexa647 antibody (magenta). The maximum intensity z-projection corresponds to a sample thickness of 750 nm. ( B ) Maximum intensity z-projection 3D-SIM image of the tubulin channel from (A) compared to the d STORM reconstruction ( C ) of the same cell. ( D ) Enlarged 3D-SIM and ( E ) d STORM images of the ROIs (dashed-line boxes) shown in ( B,C ). The d STORM image shows a direct correlation with the corresponding 3D-SIM image, but with an optical resolution of approx. 20 nm. Note that the d STORM image is obtained in HiLo mode, where in thicker parts of the cell not all of the entire volume of the cell is illuminated, resulting in small differences between the images. The single frame exposure time of the d STORM image was 20 ms and a total of 10000 frames were used for the reconstruction. The sample was mounted in Vectashield.
    Figure Legend Snippet: Correlating 3D-SIM and d STORM images of a rat liver sinusoidal endothelial cell (LSEC). ( A ) Maximum intensity z-projection 3D-SIM image of a 4-color-stained fixed rat LSEC. The nucleus was stained with DAPI (blue), actin filaments with Phalloidin-Alexa488 (green), membranes with CellMask Orange (white), and tubulin structures with anti β-tubulin mouse antibody followed by an anti-mouse IgG-Alexa647 antibody (magenta). The maximum intensity z-projection corresponds to a sample thickness of 750 nm. ( B ) Maximum intensity z-projection 3D-SIM image of the tubulin channel from (A) compared to the d STORM reconstruction ( C ) of the same cell. ( D ) Enlarged 3D-SIM and ( E ) d STORM images of the ROIs (dashed-line boxes) shown in ( B,C ). The d STORM image shows a direct correlation with the corresponding 3D-SIM image, but with an optical resolution of approx. 20 nm. Note that the d STORM image is obtained in HiLo mode, where in thicker parts of the cell not all of the entire volume of the cell is illuminated, resulting in small differences between the images. The single frame exposure time of the d STORM image was 20 ms and a total of 10000 frames were used for the reconstruction. The sample was mounted in Vectashield.

    Techniques Used: Staining, Mass Spectrometry

    3) Product Images from "Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applications"

    Article Title: Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applications

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-13-67

    Efficiency of F2A cleavage in GFP-F2A-CherryFP context. F2A sequences of various lengths were used to co-express GFP and CherryFP proteins from pGFP-F2A-CherryFP constructs ( a , schematic presentation) in vitro using TnT coupled transcription/ translation rabbit reticulocyte lysates (b) and transfected HeLa cells (c) . For TnT , reticulocyte lysates were programmed with 20 ng of plasmid DNA and translation products were resolved by the 12% SDS-PAGE. For in vivo studies, HeLa cells were transfected with 1.5 μg of plasmid DNA and harvested 30 h post transfection. Cells were lysed in RIPA buffer and equal amounts of total protein for each transfection were loaded onto 12% SDS-PAGE gel. The proteins were transferred onto a nitrocellulose membrane, blocked in PBST containing 5% milk and probed with anti-GFP (upper blot) and anti-CherryFP (lower blot) antibodies overnight at 4°C. Detection of bound primary antibody was achieved by using respective secondary antibodies, followed by ECL detection. (d) Expression of fluorescent proteins was detected in transfected HeLa cells at 24 h and 48 h post transfection in Evos microscope using 4 × objective. (e) Cellular co-localisation of [GFP-F2A] and CherryFP 30 h post-transfection. HeLa cells pre-plated on cover slips were transfected with 0.5 μg of plasmid DNA and incubated for 30 h, fixed with 100% methanol and mounted using VECTASHIELD mounting medium with DAPI. The images were acquired with Deltavision microscope using 100 × objective using Resolve 3D software. All experiments were done in triplicates.
    Figure Legend Snippet: Efficiency of F2A cleavage in GFP-F2A-CherryFP context. F2A sequences of various lengths were used to co-express GFP and CherryFP proteins from pGFP-F2A-CherryFP constructs ( a , schematic presentation) in vitro using TnT coupled transcription/ translation rabbit reticulocyte lysates (b) and transfected HeLa cells (c) . For TnT , reticulocyte lysates were programmed with 20 ng of plasmid DNA and translation products were resolved by the 12% SDS-PAGE. For in vivo studies, HeLa cells were transfected with 1.5 μg of plasmid DNA and harvested 30 h post transfection. Cells were lysed in RIPA buffer and equal amounts of total protein for each transfection were loaded onto 12% SDS-PAGE gel. The proteins were transferred onto a nitrocellulose membrane, blocked in PBST containing 5% milk and probed with anti-GFP (upper blot) and anti-CherryFP (lower blot) antibodies overnight at 4°C. Detection of bound primary antibody was achieved by using respective secondary antibodies, followed by ECL detection. (d) Expression of fluorescent proteins was detected in transfected HeLa cells at 24 h and 48 h post transfection in Evos microscope using 4 × objective. (e) Cellular co-localisation of [GFP-F2A] and CherryFP 30 h post-transfection. HeLa cells pre-plated on cover slips were transfected with 0.5 μg of plasmid DNA and incubated for 30 h, fixed with 100% methanol and mounted using VECTASHIELD mounting medium with DAPI. The images were acquired with Deltavision microscope using 100 × objective using Resolve 3D software. All experiments were done in triplicates.

    Techniques Used: Construct, In Vitro, Transfection, Plasmid Preparation, SDS Page, In Vivo, Expressing, Microscopy, Incubation, Software

    4) Product Images from "Fetal Programming by Methyl Donors Modulates Central Inflammation and Prevents Food Addiction-Like Behavior in Rats"

    Article Title: Fetal Programming by Methyl Donors Modulates Central Inflammation and Prevents Food Addiction-Like Behavior in Rats

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2020.00452

    Demethylation inhibits microglia phagocytosis in microglia cells. (A,B) Microglia cells were pre-incubated with 75 nM 5-AZA or 250 μM SAM for 24 h, or LPS followed by pre-opsonized green fluorescent latex beads incubation for 1 h at 37°C. Qualitative phagocytosis was evaluated using immunofluorescence against anti Iba-1 (1: 200) followed by goat anti-rabbit IgG coupled to Alexa Fluor 546 (1:1000) and VECTASHIELD mounting medium with DAPI. Fluorescent signals were detected by confocal-laser microscopy using an Olympus BX61W1 microscope with an FV1000 module with diode laser and ImageJ software. c p
    Figure Legend Snippet: Demethylation inhibits microglia phagocytosis in microglia cells. (A,B) Microglia cells were pre-incubated with 75 nM 5-AZA or 250 μM SAM for 24 h, or LPS followed by pre-opsonized green fluorescent latex beads incubation for 1 h at 37°C. Qualitative phagocytosis was evaluated using immunofluorescence against anti Iba-1 (1: 200) followed by goat anti-rabbit IgG coupled to Alexa Fluor 546 (1:1000) and VECTASHIELD mounting medium with DAPI. Fluorescent signals were detected by confocal-laser microscopy using an Olympus BX61W1 microscope with an FV1000 module with diode laser and ImageJ software. c p

    Techniques Used: Incubation, Immunofluorescence, Microscopy, Software

    5) Product Images from "Optimized protocol for combined PALM-dSTORM imaging"

    Article Title: Optimized protocol for combined PALM-dSTORM imaging

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-27059-z

    Comparison of the photophysical properties of overexpressed mEos2 in aqueous buffer (PBS), in the standard imaging buffer of thiols with oxygen scavenging system (MEA 100 mM + GLOX) and in the Vectashield mounting medium. ( A ) Relative number of localizations as a function of the UV laser power. A single cell was first imaged for 30 s with the 561 nm laser to bleach already activated mEos2. The same cell was then imaged using the 405 and 561 nm lasers for 30 s. Each point corresponds to number of detected localizations over 30 s for different UV laser power. All the points were normalized by the number of localizations measured during the first 30 s. ( B ) Number of detected photons per molecule per frame (integration time: 30 ms). The mode values are 500, 550 and 1250 photons for PBS, MEA + GLOX and Vectashield respectively. The median number of photons are 711, 793 and 1602 photons for PBS, MEA + GLOX and Vectashield respectively. These results ( A and B ) were obtained from the average of 5 different cells for each condition.
    Figure Legend Snippet: Comparison of the photophysical properties of overexpressed mEos2 in aqueous buffer (PBS), in the standard imaging buffer of thiols with oxygen scavenging system (MEA 100 mM + GLOX) and in the Vectashield mounting medium. ( A ) Relative number of localizations as a function of the UV laser power. A single cell was first imaged for 30 s with the 561 nm laser to bleach already activated mEos2. The same cell was then imaged using the 405 and 561 nm lasers for 30 s. Each point corresponds to number of detected localizations over 30 s for different UV laser power. All the points were normalized by the number of localizations measured during the first 30 s. ( B ) Number of detected photons per molecule per frame (integration time: 30 ms). The mode values are 500, 550 and 1250 photons for PBS, MEA + GLOX and Vectashield respectively. The median number of photons are 711, 793 and 1602 photons for PBS, MEA + GLOX and Vectashield respectively. These results ( A and B ) were obtained from the average of 5 different cells for each condition.

    Techniques Used: Imaging, Microelectrode Array, Mass Spectrometry

    6) Product Images from "Simple buffers for 3D STORM microscopy"

    Article Title: Simple buffers for 3D STORM microscopy

    Journal: Biomedical Optics Express

    doi: 10.1364/BOE.4.000885

    STORM imaging of microtubules (see section 2.2 for more details) in Vectashield. (A): Widefield image (B): Single frame, (C1): Reconstructed STORM image, with blow-up on the ROI in (C2). scale-bar = 5 μ m for (A),(B),(C1) and 1 μ m for C2.
    Figure Legend Snippet: STORM imaging of microtubules (see section 2.2 for more details) in Vectashield. (A): Widefield image (B): Single frame, (C1): Reconstructed STORM image, with blow-up on the ROI in (C2). scale-bar = 5 μ m for (A),(B),(C1) and 1 μ m for C2.

    Techniques Used: Imaging

    STORM imaging of Alexa-647 stained microtubules in a Vectashield/TRIS-Glycerol mixture: (A) 50% Vectashield and (B) 25% Vectashield. The different panels represent: (1) STORM image reconstructed from 15.000 frames, scale-bar = 500 nm (2) photon count distribution per frame and per molecule, averaged over three data-sets, and (3) standard deviation of multiple localizations giving a measure of the frame localization precision.
    Figure Legend Snippet: STORM imaging of Alexa-647 stained microtubules in a Vectashield/TRIS-Glycerol mixture: (A) 50% Vectashield and (B) 25% Vectashield. The different panels represent: (1) STORM image reconstructed from 15.000 frames, scale-bar = 500 nm (2) photon count distribution per frame and per molecule, averaged over three data-sets, and (3) standard deviation of multiple localizations giving a measure of the frame localization precision.

    Techniques Used: Imaging, Staining, Standard Deviation

    (A) Absorption spectrum of Vectashield, as well as normalized emission spectra measured at 3 different wavelengths: 400 nm (B), 560 nm (C) and 630 nm (D) with normalization factor indicated in the top right corner.
    Figure Legend Snippet: (A) Absorption spectrum of Vectashield, as well as normalized emission spectra measured at 3 different wavelengths: 400 nm (B), 560 nm (C) and 630 nm (D) with normalization factor indicated in the top right corner.

    Techniques Used:

    Statistics on STORM imaging performed in 25% Vectashield - 75% TRIS-Glycerol in which were added 1% NPG (w/v) (A), 20 mM DABCO (B), and 10 mM Lipoic Acid (C). The different panels represent: (1) photon count distribution per frame and per molecule, averaged over three datasets, (2) standard deviation of multiple localizations giving a measure of the frame localization precision, and (3) Density of molecules as a function of number of recorded frames, averaged over three measurements, with error bars indicating the standard deviation.
    Figure Legend Snippet: Statistics on STORM imaging performed in 25% Vectashield - 75% TRIS-Glycerol in which were added 1% NPG (w/v) (A), 20 mM DABCO (B), and 10 mM Lipoic Acid (C). The different panels represent: (1) photon count distribution per frame and per molecule, averaged over three datasets, (2) standard deviation of multiple localizations giving a measure of the frame localization precision, and (3) Density of molecules as a function of number of recorded frames, averaged over three measurements, with error bars indicating the standard deviation.

    Techniques Used: Imaging, Standard Deviation

    Quantifying the quality of Vectashield as a STORM buffer for Alexa-647: (A) photon count distribution per frame (blue) and per molecule (red), obtained by grouping consecutive frame localizations and (B) standard deviation of multiple localizations (see section 2.3 for grouping details), with mean values displayed in the top right corner (C) Density of molecules as a function of number of recorded frames, averaged over three measurements. The error bar indicates the standard deviation. (D) STORM image of microtubule, on which the hollowness of the structure can be resolved, as quantified in the profile taken over the 200 nm yellow-boxed region with ≈ 35 nm between the two peaks, consistent with a 25 mm structure broadened by the antibodies.
    Figure Legend Snippet: Quantifying the quality of Vectashield as a STORM buffer for Alexa-647: (A) photon count distribution per frame (blue) and per molecule (red), obtained by grouping consecutive frame localizations and (B) standard deviation of multiple localizations (see section 2.3 for grouping details), with mean values displayed in the top right corner (C) Density of molecules as a function of number of recorded frames, averaged over three measurements. The error bar indicates the standard deviation. (D) STORM image of microtubule, on which the hollowness of the structure can be resolved, as quantified in the profile taken over the 200 nm yellow-boxed region with ≈ 35 nm between the two peaks, consistent with a 25 mm structure broadened by the antibodies.

    Techniques Used: Standard Deviation

    STORM images obtained with the other working dyes (A) Alexa-555 in 20% Vectashield-80% TRIS-Glycerol (B) Cy-5 (C) CF-647 (D) Alexa-700, all in pure Vec-tashield. scale-bar = 5 μ m.
    Figure Legend Snippet: STORM images obtained with the other working dyes (A) Alexa-555 in 20% Vectashield-80% TRIS-Glycerol (B) Cy-5 (C) CF-647 (D) Alexa-700, all in pure Vec-tashield. scale-bar = 5 μ m.

    Techniques Used:

    (A) STORM image of CEP-152 stained with Cy3 using a buffer 40% Vectashield + 1% NPG + 20 mM DABCO, which improves the quality of Cy3 blinking. Scale-bar = 500 nm (B) Radial intensity distribution measured from the yellow ROI defined in (A), and Lorentzian fit showing a peak at r = 143 nm.
    Figure Legend Snippet: (A) STORM image of CEP-152 stained with Cy3 using a buffer 40% Vectashield + 1% NPG + 20 mM DABCO, which improves the quality of Cy3 blinking. Scale-bar = 500 nm (B) Radial intensity distribution measured from the yellow ROI defined in (A), and Lorentzian fit showing a peak at r = 143 nm.

    Techniques Used: Staining

    (A) Index matching with Vectashield: Optical index as a function of Vectashield concentration starting from PBS (red) or TDE (blue), and imaging performed at n = 1.5 (adapted to oil objectives) and n=1.4 (adapted to glycerol objectives) (B–D) STORM imaging of microtubules immunostained with Alexa-647 for the 25% Vectashield-75% TDE buffer and 50% Vectashield - 50% PBS buffer respectively, scale-bar = 500 nm (C–E): photon count distribution per frame and per molecule, averaged over three datasets for the 25% Vectashield-75% TDE buffer and 50% Vectashield - 50% PBS buffer respectively.
    Figure Legend Snippet: (A) Index matching with Vectashield: Optical index as a function of Vectashield concentration starting from PBS (red) or TDE (blue), and imaging performed at n = 1.5 (adapted to oil objectives) and n=1.4 (adapted to glycerol objectives) (B–D) STORM imaging of microtubules immunostained with Alexa-647 for the 25% Vectashield-75% TDE buffer and 50% Vectashield - 50% PBS buffer respectively, scale-bar = 500 nm (C–E): photon count distribution per frame and per molecule, averaged over three datasets for the 25% Vectashield-75% TDE buffer and 50% Vectashield - 50% PBS buffer respectively.

    Techniques Used: Concentration Assay, Imaging

    3D STORM of Alexa-647-labeled microtubules in Vectashield: (A) Imaging performed in 25% Vectashield-75 % TRIS-Glycerol, scale-bar = 5 μ m. (B1 2): axial profile taken from the two regions delimited in A (yellow for (B1), showing a single microtubule; red for (B2) showing two well-resolved microtubules crossing at a distance of ≈ 160 nm).
    Figure Legend Snippet: 3D STORM of Alexa-647-labeled microtubules in Vectashield: (A) Imaging performed in 25% Vectashield-75 % TRIS-Glycerol, scale-bar = 5 μ m. (B1 2): axial profile taken from the two regions delimited in A (yellow for (B1), showing a single microtubule; red for (B2) showing two well-resolved microtubules crossing at a distance of ≈ 160 nm).

    Techniques Used: Labeling, Imaging

    Related Articles

    Immunostaining:

    Article Title: Fgf10-Hippo epithelial mesenchymal crosstalk maintains and recruits lung basal stem cells
    Article Snippet: All fluorescent staining was performed with appropriate secondary antibodies from Jackson Immunoresearch, except for goat anti-chicken FITC (1:200; F-1005; Aves Labs, Inc.). .. Slides were mounted using Vectashield with (Vector Labs, H-1200) or without DAPI (Vector Labs, H-1000) depending on immunostaining. ..

    Microscopy:

    Article Title: Human Placental Trophoblasts Are Resistant to Trypanosoma cruzi Infection in a 3D-Culture Model of the Maternal-Fetal Interface
    Article Snippet: After incubation, cells were washed with PBS and stained with Alexa Fluor 488 anti-rabbit IgG (H + L) antibodies (1/1,000 dilution; Invitrogen, CA). .. Slides were mounted using Vectashield mounting medium (Vector Laboratories, Burlingame, CA) containing 4’, 6-diamindino-2-phenylindole (DAPI) and observed by microscopy. .. Phase contrast and fluorescence micrographs were acquired on a Keyene BZ-9000 fluorescence microscope at 40 or 100X magnification.

    Staining:

    Article Title: Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations
    Article Snippet: This will provide us with an initial approach allowing us to overcome one of the current limitations of super-resolution microscopy, i.e. the combination of rapid live cell imaging of cellular processes with the highest spatial resolution possible. .. Materials Reagents included Type 1 A Collagenase (Sigma Chemical, St. Louis, MO #C2674,) RPMI (Gibco Invitrogen #11875-093), CellMask Deep Red Plasma Membrane Stain, Invitrogen, #C10046 Mouse monoclonal Anti-β-Tubulin antibody, SIGMA, T8328 Alexa Fluor® 647 F(ab’)2 Fragment of Goat Anti-Mouse IgG (H + L), Invitrogen, A-21237, Lot# 1094366 CellMask Orange 5 mg/ml, Invitrogen, C10045, Lot# 1159930 Alexa Fluor 488 – phalloidin 300 u in 1.5 ml Methanol, Invitrogen, A12379, Lot# 1120408 Vybrant DiD cell-labeling solution 1 mM, Invitrogen, V22887, Lot# 1046290 VECTASHIELD Mounting Medium with DAPI, Vector Laboratories, H-1200, Collagen and Fibronectin were purchased from Sigma Chemical. .. Isolation and culture of LSECs Sprague Dawley male rats (Scanbur BK, Sollentuna, Sweden) were kept under standard conditions and fed standard chow ad libitum (Scanbur, Nittedal, Norway).

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    Vector Laboratories anti fade vectashield mounting medium
    Generation and characterization of monoclonal antibodies (mAbs) to MUC16 C-terminal (CT) domain. ( A ) Structure of MUC16 CT domain indicating the two membrane-proximal cleavage sites. The fragment used for generation of hybridomas is indicated by a line with double arrow heads. This fragment incorporates the last putative cleavage site. Other important domains are indicated: TM- Transmembrane domain; Cyt. Tail- Cytoplasmic tail. ( B ) Binding of selected anti-MUC16 CT monoclonal antibodies with purified MUC16 CT protein using an indirect ELISA. MUC16 CT protein was coated in ELISA wells and hybridoma supernatants of the indicated antibodies were added. Antibody binding was detected using a secondary antibody labeled with horseradish peroxidase and TMB substrate. ( C ) Flow cytometry analysis showing relative binding of anti-MUC16 CT mAbs (5E6 and 3H1) to MIA PaCa-2 cells transfected either with control vector or MUC16 CT FL 321 construct (last 321 amino acids of MUC16). Binding was also analyzed on OVCAR-3 (MUC16 HIGH ) and OVCAR-5 (MUC16 LOW ) cells. Anti- tandem repeat mAb M11 served as a positive control. Cells were stained with the indicated antibodies and the signal was detected using Alexa-Fluor 488 anti-mouse IgG secondary antibody. A mouse IgG1 antibody served as the irrelevant isotype control and is indicated by the gray shaded curve. ( D ) Flow cytometry analysis of OVCAR-3 cells using mAbs pre-incubated with either MUC16 peptide or irrelevant control peptide. ( E and F ) OVCAR-3 and OVCAR-5 cells were seeded on coverslips, fixed with 4% Paraformaldehyde in PBS and were either permeabilized with 0.1% Triton X-100 in PBS (E) or not permeabilized (F) and incubated with 10 μg/ml of indicated mAbs. Signal was detected using Alexa Fluor 488 conjugated secondary antibody. Coverslips were placed on glass slides containing a drop of anti-fade <t>Vectashield</t> mounting medium and observed under a ZEISS confocal laser scanning microscope (magnification, 630X).
    Anti Fade Vectashield Mounting Medium, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Generation and characterization of monoclonal antibodies (mAbs) to MUC16 C-terminal (CT) domain. ( A ) Structure of MUC16 CT domain indicating the two membrane-proximal cleavage sites. The fragment used for generation of hybridomas is indicated by a line with double arrow heads. This fragment incorporates the last putative cleavage site. Other important domains are indicated: TM- Transmembrane domain; Cyt. Tail- Cytoplasmic tail. ( B ) Binding of selected anti-MUC16 CT monoclonal antibodies with purified MUC16 CT protein using an indirect ELISA. MUC16 CT protein was coated in ELISA wells and hybridoma supernatants of the indicated antibodies were added. Antibody binding was detected using a secondary antibody labeled with horseradish peroxidase and TMB substrate. ( C ) Flow cytometry analysis showing relative binding of anti-MUC16 CT mAbs (5E6 and 3H1) to MIA PaCa-2 cells transfected either with control vector or MUC16 CT FL 321 construct (last 321 amino acids of MUC16). Binding was also analyzed on OVCAR-3 (MUC16 HIGH ) and OVCAR-5 (MUC16 LOW ) cells. Anti- tandem repeat mAb M11 served as a positive control. Cells were stained with the indicated antibodies and the signal was detected using Alexa-Fluor 488 anti-mouse IgG secondary antibody. A mouse IgG1 antibody served as the irrelevant isotype control and is indicated by the gray shaded curve. ( D ) Flow cytometry analysis of OVCAR-3 cells using mAbs pre-incubated with either MUC16 peptide or irrelevant control peptide. ( E and F ) OVCAR-3 and OVCAR-5 cells were seeded on coverslips, fixed with 4% Paraformaldehyde in PBS and were either permeabilized with 0.1% Triton X-100 in PBS (E) or not permeabilized (F) and incubated with 10 μg/ml of indicated mAbs. Signal was detected using Alexa Fluor 488 conjugated secondary antibody. Coverslips were placed on glass slides containing a drop of anti-fade Vectashield mounting medium and observed under a ZEISS confocal laser scanning microscope (magnification, 630X).

    Journal: PLoS ONE

    Article Title: Development and characterization of carboxy-terminus specific monoclonal antibodies for understanding MUC16 cleavage in human ovarian cancer

    doi: 10.1371/journal.pone.0193907

    Figure Lengend Snippet: Generation and characterization of monoclonal antibodies (mAbs) to MUC16 C-terminal (CT) domain. ( A ) Structure of MUC16 CT domain indicating the two membrane-proximal cleavage sites. The fragment used for generation of hybridomas is indicated by a line with double arrow heads. This fragment incorporates the last putative cleavage site. Other important domains are indicated: TM- Transmembrane domain; Cyt. Tail- Cytoplasmic tail. ( B ) Binding of selected anti-MUC16 CT monoclonal antibodies with purified MUC16 CT protein using an indirect ELISA. MUC16 CT protein was coated in ELISA wells and hybridoma supernatants of the indicated antibodies were added. Antibody binding was detected using a secondary antibody labeled with horseradish peroxidase and TMB substrate. ( C ) Flow cytometry analysis showing relative binding of anti-MUC16 CT mAbs (5E6 and 3H1) to MIA PaCa-2 cells transfected either with control vector or MUC16 CT FL 321 construct (last 321 amino acids of MUC16). Binding was also analyzed on OVCAR-3 (MUC16 HIGH ) and OVCAR-5 (MUC16 LOW ) cells. Anti- tandem repeat mAb M11 served as a positive control. Cells were stained with the indicated antibodies and the signal was detected using Alexa-Fluor 488 anti-mouse IgG secondary antibody. A mouse IgG1 antibody served as the irrelevant isotype control and is indicated by the gray shaded curve. ( D ) Flow cytometry analysis of OVCAR-3 cells using mAbs pre-incubated with either MUC16 peptide or irrelevant control peptide. ( E and F ) OVCAR-3 and OVCAR-5 cells were seeded on coverslips, fixed with 4% Paraformaldehyde in PBS and were either permeabilized with 0.1% Triton X-100 in PBS (E) or not permeabilized (F) and incubated with 10 μg/ml of indicated mAbs. Signal was detected using Alexa Fluor 488 conjugated secondary antibody. Coverslips were placed on glass slides containing a drop of anti-fade Vectashield mounting medium and observed under a ZEISS confocal laser scanning microscope (magnification, 630X).

    Article Snippet: Cells were washed 4 times with PBST, and mounted on glass slides in anti-fade Vectashield mounting medium containing DAPI (4′, 6-Diamidino-2-Phenylindole, Dihydrochloride) (Vector Laboratories, Burlingame, CA).

    Techniques: Binding Assay, Purification, Indirect ELISA, Enzyme-linked Immunosorbent Assay, Labeling, Flow Cytometry, Cytometry, Transfection, Plasmid Preparation, Construct, Positive Control, Staining, Incubation, Laser-Scanning Microscopy

    AML cells express less of N-cadherin and higher of vimentin proteins compared to HEK293 cells (A) Cells lysates from AML and HEK293 cells were subjected Western blot analysis. Immunoblot analysis show increased in vimentin and decreased in N-cadherin protein expression in AML cells compared to HEK293 cells. (B) AML and HEK293 cells were immunostained for vimentin and N-cadherin using double fluorescence labeling method. The cells were incubated with rabbit antibody against vimentin or N-cadherin followed by secondary anti-rabbit IgG conjugated with FITC. The cells were reacted with Vectashield Mounting Medium with Propedium Iodide (PI) for nuclear staining. (B C) FITC green signals for N-cadherin and vimentin were detected using a filter with excitation range of 488 nm and PI red signals for nuclear DNA using a filter with excitation at 535 nm. Overlay of vimentin or N-cadherin and DNA staining, demonstrating cell membrane staining for N-cadherin and cytoplasmic staining for vimentin in AML cells. To show staining specificity, control cells were stained without primary antibody. (D) Upregulation of tuberin resulted in decrease in vimentin and increase N-cadherin expression in AML cells. AML cells were infected with adenovirus 6.01 expressing tuberin complementary DNA. An adenovirus vector expressing protein (Adb-GAL) was used as a control. Immunoblot analysis shows overexpression of tuberin decreases vimentin and increases N-cadherin protein expression. Actin was used as a loading control.

    Journal: Oncotarget

    Article Title: Tuberin-deficiency downregulates N-cadherin and upregulates vimentin in kidney tumor of TSC patients

    doi:

    Figure Lengend Snippet: AML cells express less of N-cadherin and higher of vimentin proteins compared to HEK293 cells (A) Cells lysates from AML and HEK293 cells were subjected Western blot analysis. Immunoblot analysis show increased in vimentin and decreased in N-cadherin protein expression in AML cells compared to HEK293 cells. (B) AML and HEK293 cells were immunostained for vimentin and N-cadherin using double fluorescence labeling method. The cells were incubated with rabbit antibody against vimentin or N-cadherin followed by secondary anti-rabbit IgG conjugated with FITC. The cells were reacted with Vectashield Mounting Medium with Propedium Iodide (PI) for nuclear staining. (B C) FITC green signals for N-cadherin and vimentin were detected using a filter with excitation range of 488 nm and PI red signals for nuclear DNA using a filter with excitation at 535 nm. Overlay of vimentin or N-cadherin and DNA staining, demonstrating cell membrane staining for N-cadherin and cytoplasmic staining for vimentin in AML cells. To show staining specificity, control cells were stained without primary antibody. (D) Upregulation of tuberin resulted in decrease in vimentin and increase N-cadherin expression in AML cells. AML cells were infected with adenovirus 6.01 expressing tuberin complementary DNA. An adenovirus vector expressing protein (Adb-GAL) was used as a control. Immunoblot analysis shows overexpression of tuberin decreases vimentin and increases N-cadherin protein expression. Actin was used as a loading control.

    Article Snippet: The cells were reacted with Vectashield Mounting Medium with Propedium Iodide (PI) (Vector Laboratories).

    Techniques: Western Blot, Expressing, Fluorescence, Labeling, Incubation, Staining, Infection, Plasmid Preparation, Over Expression

    Fgf10-Fgfr2b signaling downstream of Yap drives secretory and basal cell amplification in the lower non-cartilaginous conducting airways upon Ilk inactivation (A) Schematic representation of non-cartilaginous airway secretory/ciliated amplification and BSC amplification or loss, stromal Fgf10 or nuclear Yap levels upon airway epithelial Ilk/Fgfr2b ablation, Ilk ablation and Ilk/Yap ablation. All lungs were collected at 2 months after tamoxifen induction. (B) Sirius red (collagen)/fast green staining and immunostaining on Sox2-Ilk f/f -Fgfr2b f/f , Sox2-Ilk f/f , and Sox2-Ilk f/f -Yap f/f non-cartilaginous airways for BSC markers p63 (red) and Keratin 5 (K5) (green). (C,D) Morphometric analysis of airway remodeling (C) and quantification of BSC numbers (#) per 100 μm basement membrane (D) from images represented in (B). (E) Relative mRNA levels of Scgb1a1 (secretory cells), FoxJ1 (ciliated cells), K5 (BSCs), p63 (BSCs) , Col1a1 (collagen) and Col3a1 (collagen) in control, Sox2-Ilk f/f (ILK KO), Sox2-Ilk f/f -Fgfr2b f/f (ILK FGFR2b DKO) and Sox2-Ilk f/f -Yap f/f (ILK YAP DKO) lungs. Nuclei were stained with DAPI (blue). ** P

    Journal: Developmental cell

    Article Title: Fgf10-Hippo epithelial mesenchymal crosstalk maintains and recruits lung basal stem cells

    doi: 10.1016/j.devcel.2017.09.003

    Figure Lengend Snippet: Fgf10-Fgfr2b signaling downstream of Yap drives secretory and basal cell amplification in the lower non-cartilaginous conducting airways upon Ilk inactivation (A) Schematic representation of non-cartilaginous airway secretory/ciliated amplification and BSC amplification or loss, stromal Fgf10 or nuclear Yap levels upon airway epithelial Ilk/Fgfr2b ablation, Ilk ablation and Ilk/Yap ablation. All lungs were collected at 2 months after tamoxifen induction. (B) Sirius red (collagen)/fast green staining and immunostaining on Sox2-Ilk f/f -Fgfr2b f/f , Sox2-Ilk f/f , and Sox2-Ilk f/f -Yap f/f non-cartilaginous airways for BSC markers p63 (red) and Keratin 5 (K5) (green). (C,D) Morphometric analysis of airway remodeling (C) and quantification of BSC numbers (#) per 100 μm basement membrane (D) from images represented in (B). (E) Relative mRNA levels of Scgb1a1 (secretory cells), FoxJ1 (ciliated cells), K5 (BSCs), p63 (BSCs) , Col1a1 (collagen) and Col3a1 (collagen) in control, Sox2-Ilk f/f (ILK KO), Sox2-Ilk f/f -Fgfr2b f/f (ILK FGFR2b DKO) and Sox2-Ilk f/f -Yap f/f (ILK YAP DKO) lungs. Nuclei were stained with DAPI (blue). ** P

    Article Snippet: Slides were mounted using Vectashield with (Vector Labs, H-1200) or without DAPI (Vector Labs, H-1000) depending on immunostaining.

    Techniques: Amplification, Staining, Immunostaining

    Inactivation of the Hippo pathway in differentiated airway epithelial cells after injury or after Ilk inactivation induces epithelial Wnt7b expression and Fgf10 secretion by ASMCs (A) Immunostaining on non-cartilaginous airways of non-injured (NI) control lungs and lungs 3 days after naphthalene (npt)-induced injury for secretory cell marker Scgb1a1 (green) or ciliated cell marker β-tubulin (green) and Yap or Merlin (red). (B) Quantification of Yap pixel intensity of pictures represented in (A) ( n ≥ 6 mice). (C) Quantification of Merlin pixel intensity of pictures represented in (A) ( n ≥ 6 mice). (D) Immunostaining on non-cartilaginous airways of control Fgf10 LacZ , Scgb1a1-Mst1/2 f/f -Fgf10 LacZ and Scgb1a1-Ilk f/f -Fgf10 LacZ lungs for secretory cell marker Scgb1a1 (green) with either Yap (red) or Wnt7b (red) or Dkk1 (red) or Fgfr2b (red) and β- gal staining of Fgf10 LacZ . Black arrowheads indicate Fgf10 expression in ASMCs. (E) Quantification of pixel intensity of Yap, Wnt7b, Dkk1 and Fgfr2b signals represented in (D) ( n ≥ 6 mice). (F) Relative mRNA expression of Wnt7b and Dkk1 in control, Scgb1a1-Mst1/2 f/f and Scgb1a1-Ilk f/f lungs. (G) Western blot analysis showing FGF10, YAP, FGFR2B, P-MST1/2, and WNT7B protein expression in control, Scgb1a1-Mst1/2 f/f (MST1/2 KO) and Scgb1a1-Ilk f/f (ILK KO) lungs. (H) Quantification of pixel intensities of pictures represented in (I) ( n ≥ 6 mice). (I) Immunostaining on control and Scgb1a1-Ilk f/f non-cartilaginous airways for Merlin (red) or phospho-Mst1/2 (red). Nuclei, DAPI (blue). ** P

    Journal: Developmental cell

    Article Title: Fgf10-Hippo epithelial mesenchymal crosstalk maintains and recruits lung basal stem cells

    doi: 10.1016/j.devcel.2017.09.003

    Figure Lengend Snippet: Inactivation of the Hippo pathway in differentiated airway epithelial cells after injury or after Ilk inactivation induces epithelial Wnt7b expression and Fgf10 secretion by ASMCs (A) Immunostaining on non-cartilaginous airways of non-injured (NI) control lungs and lungs 3 days after naphthalene (npt)-induced injury for secretory cell marker Scgb1a1 (green) or ciliated cell marker β-tubulin (green) and Yap or Merlin (red). (B) Quantification of Yap pixel intensity of pictures represented in (A) ( n ≥ 6 mice). (C) Quantification of Merlin pixel intensity of pictures represented in (A) ( n ≥ 6 mice). (D) Immunostaining on non-cartilaginous airways of control Fgf10 LacZ , Scgb1a1-Mst1/2 f/f -Fgf10 LacZ and Scgb1a1-Ilk f/f -Fgf10 LacZ lungs for secretory cell marker Scgb1a1 (green) with either Yap (red) or Wnt7b (red) or Dkk1 (red) or Fgfr2b (red) and β- gal staining of Fgf10 LacZ . Black arrowheads indicate Fgf10 expression in ASMCs. (E) Quantification of pixel intensity of Yap, Wnt7b, Dkk1 and Fgfr2b signals represented in (D) ( n ≥ 6 mice). (F) Relative mRNA expression of Wnt7b and Dkk1 in control, Scgb1a1-Mst1/2 f/f and Scgb1a1-Ilk f/f lungs. (G) Western blot analysis showing FGF10, YAP, FGFR2B, P-MST1/2, and WNT7B protein expression in control, Scgb1a1-Mst1/2 f/f (MST1/2 KO) and Scgb1a1-Ilk f/f (ILK KO) lungs. (H) Quantification of pixel intensities of pictures represented in (I) ( n ≥ 6 mice). (I) Immunostaining on control and Scgb1a1-Ilk f/f non-cartilaginous airways for Merlin (red) or phospho-Mst1/2 (red). Nuclei, DAPI (blue). ** P

    Article Snippet: Slides were mounted using Vectashield with (Vector Labs, H-1200) or without DAPI (Vector Labs, H-1000) depending on immunostaining.

    Techniques: Expressing, Immunostaining, Marker, Mouse Assay, Staining, Western Blot

    Fgf10 expression in ASMCs drives secretory and basal cell amplification in the lower non-cartilaginous conducting airways upon Ilk inactivation (A) Schematic representation of airway BSC amplification or loss, stromal Fgf10 or nuclear Yap levels in the different mutant strains. (B) Sirius red (collagen)/fast green staining and immunostaining on Scgb1a1-Ilk f/f (ILK KO), Scgb1a1-Ilk f/f -Fgfr2b f/f (ILK FGFR2b DKO) and Scgb1a1-Ilk f/f -rtTa-Dkk1 (ILK DKK1) non-cartilaginous airways for BSC markers p63 (red) and Keratin 5 (K5) (green) or secretory cell marker Scgb1a1 (green) and Yap (red). (C,D) Morphometric analysis of airway remodeling (C) and quantification of BSC numbers (D) of images represented in (B). (E) Relative mRNA levels of Scgb1a1 (secretory cells), FoxJ1 (ciliated cells), K5 (BSCs), p63 (BSCs) , Col1a1 (collagen) and Col3a1 (collagen) in control, Scgb1a1-Ilk f/f , Scgb1a1-Ilk f/f -Fgfr2b f/f and Scgb1a1-Ilk f/f -rtTa-Dkk1 lungs. Nuclei were stained with DAPI (blue). ** P

    Journal: Developmental cell

    Article Title: Fgf10-Hippo epithelial mesenchymal crosstalk maintains and recruits lung basal stem cells

    doi: 10.1016/j.devcel.2017.09.003

    Figure Lengend Snippet: Fgf10 expression in ASMCs drives secretory and basal cell amplification in the lower non-cartilaginous conducting airways upon Ilk inactivation (A) Schematic representation of airway BSC amplification or loss, stromal Fgf10 or nuclear Yap levels in the different mutant strains. (B) Sirius red (collagen)/fast green staining and immunostaining on Scgb1a1-Ilk f/f (ILK KO), Scgb1a1-Ilk f/f -Fgfr2b f/f (ILK FGFR2b DKO) and Scgb1a1-Ilk f/f -rtTa-Dkk1 (ILK DKK1) non-cartilaginous airways for BSC markers p63 (red) and Keratin 5 (K5) (green) or secretory cell marker Scgb1a1 (green) and Yap (red). (C,D) Morphometric analysis of airway remodeling (C) and quantification of BSC numbers (D) of images represented in (B). (E) Relative mRNA levels of Scgb1a1 (secretory cells), FoxJ1 (ciliated cells), K5 (BSCs), p63 (BSCs) , Col1a1 (collagen) and Col3a1 (collagen) in control, Scgb1a1-Ilk f/f , Scgb1a1-Ilk f/f -Fgfr2b f/f and Scgb1a1-Ilk f/f -rtTa-Dkk1 lungs. Nuclei were stained with DAPI (blue). ** P

    Article Snippet: Slides were mounted using Vectashield with (Vector Labs, H-1200) or without DAPI (Vector Labs, H-1000) depending on immunostaining.

    Techniques: Expressing, Amplification, Mutagenesis, Staining, Immunostaining, Marker

    The inactive Hippo pathway in basal stem/progenitor cells generates the Fgf10 -expressing tracheal stromal niche required to maintain their cell pool (A) Experimental strategy and schematic representation of tracheal BSC amplification after Fgf10 overexpression or tracheal BSC loss after Fgfr2b ablation in airway epithelial cells. Ciliated, secretory and BSCs are shown in green, blue and red, respectively. (B) Immunostaining on rtTa-Fgf10, control and Sox2-Fgfr2b f/f tracheas for the BSC markers Keratin 5 (K5) (green) and p63 (red) 14 days after doxycycline or tamoxifen induction. (C) Quantification of the number (#) of BSCs per 100 μm basement membrane of pictures represented in (B). (D) Experimental strategy and schematic representation of Mst1/2 ablation in all airway epithelial cells or selectively in secretory/ciliated cells alone or in combination with either Fgfr2b or Yap in all airway epithelial cells with or without simultaneously inducing Fgf10 expression. (E) Whole mount in situ hybridization for Fgf10 in control, Scgb1a1-Mst1/2 f/f and Sox2-Mst1/2 f/f tracheas. Note purple Fgf10 expression between the tracheal cartilage rings. (F) Upper panels show immunostaining on control, Sox2-Mst1/2 f/f and Sox2-Mst1/2 f/f -Fgfr2b f/f tracheas 2 months after tamoxifen induction as well as on a 2.5 month old Scgb1a1-Mst1/2 f/f -Confetti trachea for the BSC marker K5 (red) and the secretory cell marker Scgb1a1 (green). Lower panel shows immunostaining on Scgb1a1-Mst1/2 f/f -Confetti trachea for the BSC marker K5 (red) and GFP (green). (G) Quantification of the number (#) of BSCs per 100 μm basement membrane (BM) of pictures represented in (F,H). (H) Immunostaining on control, Sox2-Yap f/f , Sox2-Yap f/f -rtTa-Fgf10 tracheas for the BSC markers K5 (green) and p63 (red) after tamoxifen-induced deletion of Yap and/or doxycycline-induced Fgf10 expression. Refer to panel D for experimental strategy. (I) Immunostaining on adjacent tracheal sections from control, Sox2-Yap f/f and Sox2-Yap f/f -Fgf10 mice for BSC marker K5 (green) and Yap (red) or proliferation marker PCNA (red) 12 weeks after tamoxifen and 2 weeks after doxycycline induction, starting at 10 weeks after tamoxifen induction. Nuclei, DAPI (blue). ** P

    Journal: Developmental cell

    Article Title: Fgf10-Hippo epithelial mesenchymal crosstalk maintains and recruits lung basal stem cells

    doi: 10.1016/j.devcel.2017.09.003

    Figure Lengend Snippet: The inactive Hippo pathway in basal stem/progenitor cells generates the Fgf10 -expressing tracheal stromal niche required to maintain their cell pool (A) Experimental strategy and schematic representation of tracheal BSC amplification after Fgf10 overexpression or tracheal BSC loss after Fgfr2b ablation in airway epithelial cells. Ciliated, secretory and BSCs are shown in green, blue and red, respectively. (B) Immunostaining on rtTa-Fgf10, control and Sox2-Fgfr2b f/f tracheas for the BSC markers Keratin 5 (K5) (green) and p63 (red) 14 days after doxycycline or tamoxifen induction. (C) Quantification of the number (#) of BSCs per 100 μm basement membrane of pictures represented in (B). (D) Experimental strategy and schematic representation of Mst1/2 ablation in all airway epithelial cells or selectively in secretory/ciliated cells alone or in combination with either Fgfr2b or Yap in all airway epithelial cells with or without simultaneously inducing Fgf10 expression. (E) Whole mount in situ hybridization for Fgf10 in control, Scgb1a1-Mst1/2 f/f and Sox2-Mst1/2 f/f tracheas. Note purple Fgf10 expression between the tracheal cartilage rings. (F) Upper panels show immunostaining on control, Sox2-Mst1/2 f/f and Sox2-Mst1/2 f/f -Fgfr2b f/f tracheas 2 months after tamoxifen induction as well as on a 2.5 month old Scgb1a1-Mst1/2 f/f -Confetti trachea for the BSC marker K5 (red) and the secretory cell marker Scgb1a1 (green). Lower panel shows immunostaining on Scgb1a1-Mst1/2 f/f -Confetti trachea for the BSC marker K5 (red) and GFP (green). (G) Quantification of the number (#) of BSCs per 100 μm basement membrane (BM) of pictures represented in (F,H). (H) Immunostaining on control, Sox2-Yap f/f , Sox2-Yap f/f -rtTa-Fgf10 tracheas for the BSC markers K5 (green) and p63 (red) after tamoxifen-induced deletion of Yap and/or doxycycline-induced Fgf10 expression. Refer to panel D for experimental strategy. (I) Immunostaining on adjacent tracheal sections from control, Sox2-Yap f/f and Sox2-Yap f/f -Fgf10 mice for BSC marker K5 (green) and Yap (red) or proliferation marker PCNA (red) 12 weeks after tamoxifen and 2 weeks after doxycycline induction, starting at 10 weeks after tamoxifen induction. Nuclei, DAPI (blue). ** P

    Article Snippet: Slides were mounted using Vectashield with (Vector Labs, H-1200) or without DAPI (Vector Labs, H-1000) depending on immunostaining.

    Techniques: Expressing, Amplification, Over Expression, Immunostaining, In Situ Hybridization, Marker, Mouse Assay

    Demethylation inhibits microglia phagocytosis in microglia cells. (A,B) Microglia cells were pre-incubated with 75 nM 5-AZA or 250 μM SAM for 24 h, or LPS followed by pre-opsonized green fluorescent latex beads incubation for 1 h at 37°C. Qualitative phagocytosis was evaluated using immunofluorescence against anti Iba-1 (1: 200) followed by goat anti-rabbit IgG coupled to Alexa Fluor 546 (1:1000) and VECTASHIELD mounting medium with DAPI. Fluorescent signals were detected by confocal-laser microscopy using an Olympus BX61W1 microscope with an FV1000 module with diode laser and ImageJ software. c p

    Journal: Frontiers in Neuroscience

    Article Title: Fetal Programming by Methyl Donors Modulates Central Inflammation and Prevents Food Addiction-Like Behavior in Rats

    doi: 10.3389/fnins.2020.00452

    Figure Lengend Snippet: Demethylation inhibits microglia phagocytosis in microglia cells. (A,B) Microglia cells were pre-incubated with 75 nM 5-AZA or 250 μM SAM for 24 h, or LPS followed by pre-opsonized green fluorescent latex beads incubation for 1 h at 37°C. Qualitative phagocytosis was evaluated using immunofluorescence against anti Iba-1 (1: 200) followed by goat anti-rabbit IgG coupled to Alexa Fluor 546 (1:1000) and VECTASHIELD mounting medium with DAPI. Fluorescent signals were detected by confocal-laser microscopy using an Olympus BX61W1 microscope with an FV1000 module with diode laser and ImageJ software. c p

    Article Snippet: Finally, cells were washed 3 × with 1 × PBS and mounted in VECTASHIELD mounting medium with DAPI (Vector Laboratories, H-1000-10).

    Techniques: Incubation, Immunofluorescence, Microscopy, Software