donkey anti rabbit igg h l highly cross adsorbed secondary antibody  (Thermo Fisher)


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    Name:
    Donkey anti Rabbit IgG H L Highly Cross Adsorbed Secondary Antibody
    Description:
    Donkey anti Rabbit IgG H L Highly Cross Adsorbed Secondary Antibody for IF ICC IHC Flow
    Catalog Number:
    A10039
    Price:
    None
    Category:
    Antibodies Secondary Detection Reagents
    Applications:
    Antibodies and Secondary Detection|Cell Analysis|Secondary Detection
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    Structured Review

    Thermo Fisher donkey anti rabbit igg h l highly cross adsorbed secondary antibody
    Donkey anti Rabbit IgG H L Highly Cross Adsorbed Secondary Antibody for IF ICC IHC Flow
    https://www.bioz.com/result/donkey anti rabbit igg h l highly cross adsorbed secondary antibody/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    donkey anti rabbit igg h l highly cross adsorbed secondary antibody - by Bioz Stars, 2021-04
    99/100 stars

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    Related Articles

    Incubation:

    Article Title: Neurodegeneration and Unfolded-Protein Response in Mice Expressing a Membrane-Tethered Flexible Tail of PrP
    Article Snippet: Cells were rinsed 1x with PBS and incubated with primary antibody (POM11, giantin, LAMP2) diluted in washing buffer (1%BSA, 0.25% Triton X-100 in PBS) overnight at 4°C. .. After washing 2X with PBS, cells were incubated with Alexa Fluor-conjugated 555- anti-mouse or 647-secondary anti-rabbit or anti-rat antibodies (Invitrogen, Molecular Probes) diluted 1:5000 in washing buffer for 2h at RT, and incubated with 1 ug/ml DAPI (4’,6-diamidino-2-phenylindole, Invitrogen). ..

    Article Title: Optical Clearing in the Kidney Reveals Potassium-Mediated Tubule Remodeling
    Article Snippet: Samples were washed with 1× wash buffer overnight at room temperature, then placed in Smart Clear II Pro (Lifecanvas Technologies) for 3 days (kidney slices) or 7 days (whole kidney). .. Samples were washed with 1× wash buffer overnight at room temperature, then immunofluorescence protocol was performed as described in the ethyl cinnamate section with the exception that we did not perform antigen retrieval, and tissue was stained with pThr53-NCC (1:100) or propidium iodide (PI, 1:1000, Life Technologies, Cat#P3566). pThr53-NCC-stained samples were washed overnight and then incubated with donkey anti–rabbit Cy5 (1:200; Life Technologies, CatA31573) for 4 days. .. Imaging was performed after at least 24hrs of incubation in Easy Index clearing solution (Lifecanvas Technologies, Cat#EI-Z1001).

    other:

    Article Title: Paired immunoglobulin-like receptor B regulates platelet activation
    Article Snippet: The Alexa 488-conjugated Fg, Alexa 488-conjugated phalloidin, rhodamine-conjugated phalloidin, Alexa 594-conjagated donkey anti-goat antibody, and Alexa 488-conjugated donkey anti-rabbit antibody were from Life Technologies (Gaithersburg, MD).

    Transfection:

    Article Title: Activation of the Ca2+ sensing receptor and the PKC/WNK4 downstream signaling cascade induces incorporation of ZO-2 to tight junctions and its separation from 14-3-3
    Article Snippet: In these experiments, cells transfected with HA-ZO-2 WT or the mutants HA-ZO-2 S261A and HA-ZO-2 T248A were identified employing the same rabbit antibody against HA followed by a secondary donkey IgG anti rabbit coupled to Alexa Fluor 488 (Cat. A21206, dilution 1:500, Life Technologies, Carlsbad, CA) ( ); and 7) a rabbit antibody against Flag (Cat. Sc-807, dilution 1:100, Santa Cruz Biotechnology, Dallas, TX) and a mouse antibody against HA (Cat 26183-D800, dilution 1:100, Life Technologies, Carlsbad, CA). .. In this experiment the cells transfected with HA-ZO-2 WT or the mutants HA-ZO-2 S261A and HA-ZO-2 T248A were identified employing the same rabbit antibody against HA followed by a secondary donkey IgG anti rabbit coupled to Alexa Fluor 488 (Cat. A21206, dilution 1:500, Life Technologies, Carlsbad, CA) ( ). ..

    Immunofluorescence:

    Article Title: Optical Clearing in the Kidney Reveals Potassium-Mediated Tubule Remodeling
    Article Snippet: Samples were washed with 1× wash buffer overnight at room temperature, then placed in Smart Clear II Pro (Lifecanvas Technologies) for 3 days (kidney slices) or 7 days (whole kidney). .. Samples were washed with 1× wash buffer overnight at room temperature, then immunofluorescence protocol was performed as described in the ethyl cinnamate section with the exception that we did not perform antigen retrieval, and tissue was stained with pThr53-NCC (1:100) or propidium iodide (PI, 1:1000, Life Technologies, Cat#P3566). pThr53-NCC-stained samples were washed overnight and then incubated with donkey anti–rabbit Cy5 (1:200; Life Technologies, CatA31573) for 4 days. .. Imaging was performed after at least 24hrs of incubation in Easy Index clearing solution (Lifecanvas Technologies, Cat#EI-Z1001).

    Article Title: Regulation of cargo transfer between ESCRT-0 and ESCRT-I complexes by flotillin-1 during endosomal sorting of ubiquitinated cargo
    Article Snippet: The rabbit polyclonal anti-GFP (cat. no. 632592; IP: 1/300) was from Takara Bio (Mountain View, CA, USA). .. For immunofluorescence, primary antibodies were detected with Cy3- or Cy5-conjugated goat anti-mouse or anti-rabbit antibodies (Jackson Immunoresearch, West Grove, PA, USA) or with Alexa Fluor 488, 546 or 647 donkey anti-rabbit or anti-mouse antibodies (Molecular Probes, Karlsruhe, Germany). .. Rat flotillin-1 and -2 coding regions were cloned into pET-41a vector for bacterial expression as GST fusions (Merck/Millipore, Darmstadt, Germany).

    Staining:

    Article Title: Optical Clearing in the Kidney Reveals Potassium-Mediated Tubule Remodeling
    Article Snippet: Samples were washed with 1× wash buffer overnight at room temperature, then placed in Smart Clear II Pro (Lifecanvas Technologies) for 3 days (kidney slices) or 7 days (whole kidney). .. Samples were washed with 1× wash buffer overnight at room temperature, then immunofluorescence protocol was performed as described in the ethyl cinnamate section with the exception that we did not perform antigen retrieval, and tissue was stained with pThr53-NCC (1:100) or propidium iodide (PI, 1:1000, Life Technologies, Cat#P3566). pThr53-NCC-stained samples were washed overnight and then incubated with donkey anti–rabbit Cy5 (1:200; Life Technologies, CatA31573) for 4 days. .. Imaging was performed after at least 24hrs of incubation in Easy Index clearing solution (Lifecanvas Technologies, Cat#EI-Z1001).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Optical Clearing in the Kidney Reveals Potassium-Mediated Tubule Remodeling
    Article Snippet: Samples were washed with 1× wash buffer overnight at room temperature, then placed in Smart Clear II Pro (Lifecanvas Technologies) for 3 days (kidney slices) or 7 days (whole kidney). .. Samples were washed with 1× wash buffer overnight at room temperature, then immunofluorescence protocol was performed as described in the ethyl cinnamate section with the exception that we did not perform antigen retrieval, and tissue was stained with pThr53-NCC (1:100) or propidium iodide (PI, 1:1000, Life Technologies, Cat#P3566). pThr53-NCC-stained samples were washed overnight and then incubated with donkey anti–rabbit Cy5 (1:200; Life Technologies, CatA31573) for 4 days. .. Imaging was performed after at least 24hrs of incubation in Easy Index clearing solution (Lifecanvas Technologies, Cat#EI-Z1001).

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  • 97
    Thermo Fisher 594 labeled donkey anti rabbit immunoglobulin
    Transfection of I/1Ki and Vero E6 cells with pCAGGS-SDeV-FWD and pCAGGS-SDeV-REV constructs results in SDeV replication. (A) I/1Ki (top) and Vero E6 (bottom) cells transfected with Δ-fwd (pCAGGS-SDeV-FWD) and Δ-rev (pCAGGS-SDeV-REV) were stained for SDAg (anti-SDAg antiserum [1:7,500] and Alexa Fluor <t>594-labeled</t> donkey anti-rabbit immunoglobulin [1:1,000]) at 1, 2, 3, and 4 days posttransfection (from left to right). The images were taken at ×400 magnification using a Zeiss Axioplan 2 microscope. (B) Western blot of I/1Ki (left panel) and Vero E6 (right panel) cell pellets at 1, 2, 3, and 4 days posttransfection with Δ-fwd and Δ-rev constructs. Precision Plus Protein Dual Color Standards (Bio-Rad) served as the marker, and the results were recorded using the Odyssey infrared imaging system (Li-Cor).
    594 Labeled Donkey Anti Rabbit Immunoglobulin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/594 labeled donkey anti rabbit immunoglobulin/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    594 labeled donkey anti rabbit immunoglobulin - by Bioz Stars, 2021-04
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    Transfection of I/1Ki and Vero E6 cells with pCAGGS-SDeV-FWD and pCAGGS-SDeV-REV constructs results in SDeV replication. (A) I/1Ki (top) and Vero E6 (bottom) cells transfected with Δ-fwd (pCAGGS-SDeV-FWD) and Δ-rev (pCAGGS-SDeV-REV) were stained for SDAg (anti-SDAg antiserum [1:7,500] and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin [1:1,000]) at 1, 2, 3, and 4 days posttransfection (from left to right). The images were taken at ×400 magnification using a Zeiss Axioplan 2 microscope. (B) Western blot of I/1Ki (left panel) and Vero E6 (right panel) cell pellets at 1, 2, 3, and 4 days posttransfection with Δ-fwd and Δ-rev constructs. Precision Plus Protein Dual Color Standards (Bio-Rad) served as the marker, and the results were recorded using the Odyssey infrared imaging system (Li-Cor).

    Journal: mBio

    Article Title: Snake Deltavirus Utilizes Envelope Proteins of Different Viruses To Generate Infectious Particles

    doi: 10.1128/mBio.03250-19

    Figure Lengend Snippet: Transfection of I/1Ki and Vero E6 cells with pCAGGS-SDeV-FWD and pCAGGS-SDeV-REV constructs results in SDeV replication. (A) I/1Ki (top) and Vero E6 (bottom) cells transfected with Δ-fwd (pCAGGS-SDeV-FWD) and Δ-rev (pCAGGS-SDeV-REV) were stained for SDAg (anti-SDAg antiserum [1:7,500] and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin [1:1,000]) at 1, 2, 3, and 4 days posttransfection (from left to right). The images were taken at ×400 magnification using a Zeiss Axioplan 2 microscope. (B) Western blot of I/1Ki (left panel) and Vero E6 (right panel) cell pellets at 1, 2, 3, and 4 days posttransfection with Δ-fwd and Δ-rev constructs. Precision Plus Protein Dual Color Standards (Bio-Rad) served as the marker, and the results were recorded using the Odyssey infrared imaging system (Li-Cor).

    Article Snippet: For secondary antibodies, we used the following dilutions: 1:1,000 for Alexa Fluor 488- or 594-labeled donkey anti-rabbit immunoglobulin (ThermoFisher Scientific) and 1:1,000 for Alexa Fluor 488- or 594-labeled donkey anti-mouse immunoglobulin (ThermoFisher Scientific).

    Techniques: Transfection, Construct, Staining, Labeling, Microscopy, Western Blot, Marker, Imaging

    SDeV-infected cells can be superinfected with reptarenaviruses (UHV-2 and UGV-1) and hartmanivirus (HISV-1). (A) Mock-infected I/1Ki cells (boa kidney) and mock-, UGV-1-, and UHV-2-infected I/1Ki-Δ cells were stained for SDAg (anti-SDAg-AF488 [α-SDAg-AF488], left panels, green) and reptarenavirus NP (α-NP-AF594, second column, red). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the three images. (B) Mock-, UGV-1-, and HISV-1-infected V/1Ki-Δ cells (boa kidney) were stained for SDAg (α-SDAg-AF488, left panels, green), reptarenavirus NP (α-NP-AF594, second column, except bottom, red), or hartmanivirus NP (anti-HISV NP [1:3,000] and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin [1:1,000], second column bottom panel, red). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the three images. (C) Mock-, UGV-1-, and HISV-1-infected V/1Liv-Δ cells (boa liver) were stained for SDAg (α-SDAg-AF488, left panels, green), reptarenavirus NP (α-NP-AF594, second column, except bottom, red), or hartmanivirus NP (anti-HISV NP [1:3,000] and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin [1:1,000], second column bottom panel, red). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the three images. (D) Mock-, UGV-1-, and HISV-1-infected V/2Hz-Δ cells (boa heart) were stained for SDAg (α-SDAg-AF488, left panels, green), reptarenavirus NP (α-NP-AF594, second column, except bottom, red), or hartmanivirus NP (anti-HISV NP [1:3,000] and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin [1:1,000], second column bottom panel, red). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the three images. All images were taken at ×400 magnification using a Zeiss Axioplan 2 microscope, and a 30-μm bar is shown in the bottom right corner of each panel.

    Journal: mBio

    Article Title: Snake Deltavirus Utilizes Envelope Proteins of Different Viruses To Generate Infectious Particles

    doi: 10.1128/mBio.03250-19

    Figure Lengend Snippet: SDeV-infected cells can be superinfected with reptarenaviruses (UHV-2 and UGV-1) and hartmanivirus (HISV-1). (A) Mock-infected I/1Ki cells (boa kidney) and mock-, UGV-1-, and UHV-2-infected I/1Ki-Δ cells were stained for SDAg (anti-SDAg-AF488 [α-SDAg-AF488], left panels, green) and reptarenavirus NP (α-NP-AF594, second column, red). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the three images. (B) Mock-, UGV-1-, and HISV-1-infected V/1Ki-Δ cells (boa kidney) were stained for SDAg (α-SDAg-AF488, left panels, green), reptarenavirus NP (α-NP-AF594, second column, except bottom, red), or hartmanivirus NP (anti-HISV NP [1:3,000] and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin [1:1,000], second column bottom panel, red). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the three images. (C) Mock-, UGV-1-, and HISV-1-infected V/1Liv-Δ cells (boa liver) were stained for SDAg (α-SDAg-AF488, left panels, green), reptarenavirus NP (α-NP-AF594, second column, except bottom, red), or hartmanivirus NP (anti-HISV NP [1:3,000] and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin [1:1,000], second column bottom panel, red). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the three images. (D) Mock-, UGV-1-, and HISV-1-infected V/2Hz-Δ cells (boa heart) were stained for SDAg (α-SDAg-AF488, left panels, green), reptarenavirus NP (α-NP-AF594, second column, except bottom, red), or hartmanivirus NP (anti-HISV NP [1:3,000] and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin [1:1,000], second column bottom panel, red). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the three images. All images were taken at ×400 magnification using a Zeiss Axioplan 2 microscope, and a 30-μm bar is shown in the bottom right corner of each panel.

    Article Snippet: For secondary antibodies, we used the following dilutions: 1:1,000 for Alexa Fluor 488- or 594-labeled donkey anti-rabbit immunoglobulin (ThermoFisher Scientific) and 1:1,000 for Alexa Fluor 488- or 594-labeled donkey anti-mouse immunoglobulin (ThermoFisher Scientific).

    Techniques: Infection, Staining, Labeling, Microscopy

    Infectious SDeV particles are formed when I/1Ki-Δ cells are transfected with viral glycoproteins. (A) I/1Ki-Δ cells transfected with HISV-1 GPC (top row), Puumala virus glycoproteins (PUUV Gn Gc, second row), UGV-1 GPC (third row), UGV-1 ZP and GPC (fourth row), HBV S-Ag (fifth row), and empty pCAGGS-MCS plasmid (bottom row) were stained for HA tag (anti-HA [1:4,000] and Alexa Fluor 594-labeled donkey anti-mouse immunoglobulin [1:1,000], left panels, red) and SDAg (α-SDAg-AF488, middle panels, green). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the two images. A 30-μm bar is shown in the bottom right corner. All images were taken at ×400 magnification using a Zeiss Axioplan 2 microscope. (B) Supernatants collected from I/1Ki-Δ cells transfected with empty pCAGGS-MCS plasmid, UGV-1 ZP, UGV-1 GPC and ZP, UGV-1 GPC, HISV-1 GPC, LCMV GPC, JUNV GPC, PUUV glycoproteins, and HBV S-Ag were pelleted by ultracentrifugation and analyzed by Western blotting. The left panel shows anti-SDAg staining, and the right panel shows anti-SDAg and anti-HA staining. (C) Supernatants collected from I/1Ki-Δ cells transfected with empty pCAGGS-MCS plasmid, UGV-1 ZP, UGV-1 GPC and ZP, UGV-1 GPC, HISV-1 GPC, LCMV GPC, JUNV GPC, PUUV glycoproteins, and HBV S-Ag were titrated on clean I/1Ki cells. The plasmid used for transfection is shown in the left column, and the corresponding SDeV titer (in fluorescent focus-forming units [fffus] per milliliter) is shown in the right column.

    Journal: mBio

    Article Title: Snake Deltavirus Utilizes Envelope Proteins of Different Viruses To Generate Infectious Particles

    doi: 10.1128/mBio.03250-19

    Figure Lengend Snippet: Infectious SDeV particles are formed when I/1Ki-Δ cells are transfected with viral glycoproteins. (A) I/1Ki-Δ cells transfected with HISV-1 GPC (top row), Puumala virus glycoproteins (PUUV Gn Gc, second row), UGV-1 GPC (third row), UGV-1 ZP and GPC (fourth row), HBV S-Ag (fifth row), and empty pCAGGS-MCS plasmid (bottom row) were stained for HA tag (anti-HA [1:4,000] and Alexa Fluor 594-labeled donkey anti-mouse immunoglobulin [1:1,000], left panels, red) and SDAg (α-SDAg-AF488, middle panels, green). Hoechst 33342 was used to visualize the nuclei. The panels on the right show an overlay of the two images. A 30-μm bar is shown in the bottom right corner. All images were taken at ×400 magnification using a Zeiss Axioplan 2 microscope. (B) Supernatants collected from I/1Ki-Δ cells transfected with empty pCAGGS-MCS plasmid, UGV-1 ZP, UGV-1 GPC and ZP, UGV-1 GPC, HISV-1 GPC, LCMV GPC, JUNV GPC, PUUV glycoproteins, and HBV S-Ag were pelleted by ultracentrifugation and analyzed by Western blotting. The left panel shows anti-SDAg staining, and the right panel shows anti-SDAg and anti-HA staining. (C) Supernatants collected from I/1Ki-Δ cells transfected with empty pCAGGS-MCS plasmid, UGV-1 ZP, UGV-1 GPC and ZP, UGV-1 GPC, HISV-1 GPC, LCMV GPC, JUNV GPC, PUUV glycoproteins, and HBV S-Ag were titrated on clean I/1Ki cells. The plasmid used for transfection is shown in the left column, and the corresponding SDeV titer (in fluorescent focus-forming units [fffus] per milliliter) is shown in the right column.

    Article Snippet: For secondary antibodies, we used the following dilutions: 1:1,000 for Alexa Fluor 488- or 594-labeled donkey anti-rabbit immunoglobulin (ThermoFisher Scientific) and 1:1,000 for Alexa Fluor 488- or 594-labeled donkey anti-mouse immunoglobulin (ThermoFisher Scientific).

    Techniques: Transfection, Gel Permeation Chromatography, Plasmid Preparation, Staining, Labeling, Microscopy, Western Blot

    Binding of HAE-1, HAE-4, and control antibody to human apoE4 in the brains of living mice. ( A ) Control IgG2ab ( n = 7), HAE-1 ( n = 5), and HAE-4 ( n = 6) conjugated with Alexa 594 were applied directly onto the surface of the brain in living APPPS1-21/APOE4 mice that were 6 months of age, and antibody localization was observed using 2-photon microscopy. Amyloid was labeled using methoxy-X04. The signal from Alexa 594 and methoxy-X04 was merged (MERGE) to show the colocalization of antibodies and plaques. ( B ) Control human IgG ( n = 2) or chi–HAE-4 at 50 mg/kg body weight was injected i.p. in 1 dose (0 hour, n = 3) or 2 doses (0 and 48 hour, n = 3). APPPS1-21/APOE4 mice were sacrificed 48 hours after final injection. The antibodies in the brain were detected by biotinylated rabbit anti–human IgG followed by DAB. Left panel, bar = 1 mm. Right panel, high-power image of the indicated areas shown in the left panel; bar = 300 μm.

    Journal: The Journal of Clinical Investigation

    Article Title: Targeting of nonlipidated, aggregated apoE with antibodies inhibits amyloid accumulation

    doi: 10.1172/JCI96429

    Figure Lengend Snippet: Binding of HAE-1, HAE-4, and control antibody to human apoE4 in the brains of living mice. ( A ) Control IgG2ab ( n = 7), HAE-1 ( n = 5), and HAE-4 ( n = 6) conjugated with Alexa 594 were applied directly onto the surface of the brain in living APPPS1-21/APOE4 mice that were 6 months of age, and antibody localization was observed using 2-photon microscopy. Amyloid was labeled using methoxy-X04. The signal from Alexa 594 and methoxy-X04 was merged (MERGE) to show the colocalization of antibodies and plaques. ( B ) Control human IgG ( n = 2) or chi–HAE-4 at 50 mg/kg body weight was injected i.p. in 1 dose (0 hour, n = 3) or 2 doses (0 and 48 hour, n = 3). APPPS1-21/APOE4 mice were sacrificed 48 hours after final injection. The antibodies in the brain were detected by biotinylated rabbit anti–human IgG followed by DAB. Left panel, bar = 1 mm. Right panel, high-power image of the indicated areas shown in the left panel; bar = 300 μm.

    Article Snippet: Iba-1+ cells were detected using rabbit anti–Iba-1 (catalog 019-19741, Wako) followed by donkey anti–rabbit IgG Alexa Fluor 647 (catalog A-31573, ThermoFisher Scientific).

    Techniques: Binding Assay, Mouse Assay, Microscopy, Labeling, Injection

    PKCε activation increases the interaction of ZO-2 with WNK4 in monolayers cultured in LC media. (A) PLA assays done in monolayers cultured in LC or after a CS with a rabbit antibody against ZO-2 and a mouse antibody anti-HA. Monolayers were treated or not for 2 h with 0.5 mM DiC8 or 200 nM bryostatin. In addition, as indicated, some monolayers were pretreated for 30 min and thereafter for 2 h with 25 nM Ro-318220, or 2 μM of the PKCε inhibitor permeable peptide εv1-2. Cells transfected with WNK4-HA were identified with a mouse antibody anti HA, followed by a secondary goat anti-mouse IgG coupled to Alexa Fluor 488. Background corresponds to LC cultured cells not transfected with WNK4-HA. Bars, 25 μm. Top panel, representative images; bottom panel, quantitative analysis done using BlobFinder. Statistical analysis was done with Kruskal-Wallis test followed by Dunn’s multiple comparison test, * p

    Journal: Molecular Biology of the Cell

    Article Title: Activation of the Ca2+ sensing receptor and the PKC/WNK4 downstream signaling cascade induces incorporation of ZO-2 to tight junctions and its separation from 14-3-3

    doi: 10.1091/mbc.E18-09-0591

    Figure Lengend Snippet: PKCε activation increases the interaction of ZO-2 with WNK4 in monolayers cultured in LC media. (A) PLA assays done in monolayers cultured in LC or after a CS with a rabbit antibody against ZO-2 and a mouse antibody anti-HA. Monolayers were treated or not for 2 h with 0.5 mM DiC8 or 200 nM bryostatin. In addition, as indicated, some monolayers were pretreated for 30 min and thereafter for 2 h with 25 nM Ro-318220, or 2 μM of the PKCε inhibitor permeable peptide εv1-2. Cells transfected with WNK4-HA were identified with a mouse antibody anti HA, followed by a secondary goat anti-mouse IgG coupled to Alexa Fluor 488. Background corresponds to LC cultured cells not transfected with WNK4-HA. Bars, 25 μm. Top panel, representative images; bottom panel, quantitative analysis done using BlobFinder. Statistical analysis was done with Kruskal-Wallis test followed by Dunn’s multiple comparison test, * p

    Article Snippet: In this experiment the cells transfected with HA-ZO-2 WT or the mutants HA-ZO-2 S261A and HA-ZO-2 T248A were identified employing the same rabbit antibody against HA followed by a secondary donkey IgG anti rabbit coupled to Alexa Fluor 488 (Cat. A21206, dilution 1:500, Life Technologies, Carlsbad, CA) ( ).

    Techniques: Activation Assay, Cell Culture, Proximity Ligation Assay, Transfection

    BrdU + Cells in AQP2 + Tubules (A) AQP2 + tubules in CLARITY-cleared control kidney. Scale bars, 1 mm. (B) AQP2 + tubules in control kidney. The CLARITY protocol was first applied, and then tissue was washed, dehydrated, and cleared with ethyl cinnamate. Scale bar, 200 μm. (C–D) Ethyl cinnamate-cleared kidney slices from mice on 3-day normal (C) or low (D) potassium diet were co-stained for AQP2 and BrdU. (E–G) Data analysis process to determine BrdU + cells within the AQP2 + medullary collecting duct in mice on low potassium diet. (H) Quantification of BrdU + cells within the AQP2 + medullary collecting duct in mice on normal (C) or low (D) normal and low-potassium diets. Two kidney slices per sample were used, and from each kidney slice, five z stacks were taken from the outer medulla (covering ~50% of the outer stripe [OSOM] and 50% of the inner stripe [ISOM]). Imaging was performed with confocal microscopy using a 10× objective lens (Zeiss LSM 880 with Airyscan). Data are represented as mean ± SEM (n = 3 mice). p values were calculated using an unpaired two-tailed Student’s t test. (I and J) A representative z stack shows an absence of nonspecific binding of secondary antibody (I, donkey anti-mouse Cy5 [pseudocolor: red]; J, donkey anti-goat Cy3 [pseudocolor: green]) without previous incubation in primary antibody. Scale bars, 100 μm. .

    Journal: Cell reports

    Article Title: Optical Clearing in the Kidney Reveals Potassium-Mediated Tubule Remodeling

    doi: 10.1016/j.celrep.2018.11.021

    Figure Lengend Snippet: BrdU + Cells in AQP2 + Tubules (A) AQP2 + tubules in CLARITY-cleared control kidney. Scale bars, 1 mm. (B) AQP2 + tubules in control kidney. The CLARITY protocol was first applied, and then tissue was washed, dehydrated, and cleared with ethyl cinnamate. Scale bar, 200 μm. (C–D) Ethyl cinnamate-cleared kidney slices from mice on 3-day normal (C) or low (D) potassium diet were co-stained for AQP2 and BrdU. (E–G) Data analysis process to determine BrdU + cells within the AQP2 + medullary collecting duct in mice on low potassium diet. (H) Quantification of BrdU + cells within the AQP2 + medullary collecting duct in mice on normal (C) or low (D) normal and low-potassium diets. Two kidney slices per sample were used, and from each kidney slice, five z stacks were taken from the outer medulla (covering ~50% of the outer stripe [OSOM] and 50% of the inner stripe [ISOM]). Imaging was performed with confocal microscopy using a 10× objective lens (Zeiss LSM 880 with Airyscan). Data are represented as mean ± SEM (n = 3 mice). p values were calculated using an unpaired two-tailed Student’s t test. (I and J) A representative z stack shows an absence of nonspecific binding of secondary antibody (I, donkey anti-mouse Cy5 [pseudocolor: red]; J, donkey anti-goat Cy3 [pseudocolor: green]) without previous incubation in primary antibody. Scale bars, 100 μm. .

    Article Snippet: Samples were washed with 1× wash buffer overnight at room temperature, then immunofluorescence protocol was performed as described in the ethyl cinnamate section with the exception that we did not perform antigen retrieval, and tissue was stained with pThr53-NCC (1:100) or propidium iodide (PI, 1:1000, Life Technologies, Cat#P3566). pThr53-NCC-stained samples were washed overnight and then incubated with donkey anti–rabbit Cy5 (1:200; Life Technologies, Cat#A31573) for 4 days.

    Techniques: Mouse Assay, Staining, Imaging, Confocal Microscopy, Two Tailed Test, Binding Assay, Incubation