alexa fluor 647 conjugated anti rabbit  (Jackson Immuno)

 
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    Name:
    Alexa Fluor 488 AffiniPure F ab ₂ Fragment Donkey Anti Mouse IgG
    Description:
    F ab 2 fragment antibodies are generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving some of the hinge region F ab 2 fragments have two antigen binding Fab portions linked together by disulfide bonds and therefore they are divalent The average molecular weight is about 110 kDa They are used for specific applications such as to avoid binding of secondary antibodies to live cells with Fc receptors or to Protein A or Protein G Based on immunoelectrophoresis and or ELISA the antibody reacts with whole molecule mouse IgG It also reacts with the light chains of other mouse immunoglobulins No antibody was detected against non immunoglobulin serum proteins The antibody has been tested by ELISA and or solid phase adsorbed to ensure minimal cross reaction with bovine chicken goat guinea pig syrian hamster horse human rabbit and sheep serum proteins but it may cross react with immunoglobulins from other species
    Catalog Number:
    715-546-150
    Price:
    180.0
    Category:
    F ab ₂ Fragment Affinity Purified Antibodies
    Conjugate:
    Alexa Fluor 488
    Size:
    0 3 mg
    Format:
    F(ab')₂ Fragment
    Host:
    Donkey
    Buy from Supplier


    Structured Review

    Jackson Immuno alexa fluor 647 conjugated anti rabbit
    TCN induces translocation of nuclear HuR to cytoplasm in an ERK-dependent manner. ( a ) HepG2 cells were treated with, or without U0126 for one hour prior to addition of TCN or not for four hours and total lysate were immunoblotted with the indicated antibodies. ( b ) The cells were treated with TCN for four hours and subjected to subcellular fractionation with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents. Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. ( c ) Cells were treated with, or without, U0126 for one hour prior to the addition of TCN for four hours and subjected to subcellular fractionation and the cytosolic fraction was immunoblotted with the indicated antibodies. ( d ) HA-HuR transfected cells were exposed to TCN or PMA for four hours and further examined by confocal immunofluorescence microscopy with anti-HA. ( e ) Merged picture of HA-HuR and nuclei (Hoechst) in cells treated as described in ( d ) and with an increased resolution compared to ( d ). Red: HA-HuR, <t>Alexa</t> fluor 647, blue: nuclei, Hoechst. One representative experiment is shown ( n = 3). ( f ) Nuclear and cytoplasmic HA-HuR was localized in 600–900 cells for each treatment. The proportion of cells with nuclear and cytoplasmic HA-HuR, respectively, to the total number of cells counted, are shown ( n = 1).
    F ab 2 fragment antibodies are generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving some of the hinge region F ab 2 fragments have two antigen binding Fab portions linked together by disulfide bonds and therefore they are divalent The average molecular weight is about 110 kDa They are used for specific applications such as to avoid binding of secondary antibodies to live cells with Fc receptors or to Protein A or Protein G Based on immunoelectrophoresis and or ELISA the antibody reacts with whole molecule mouse IgG It also reacts with the light chains of other mouse immunoglobulins No antibody was detected against non immunoglobulin serum proteins The antibody has been tested by ELISA and or solid phase adsorbed to ensure minimal cross reaction with bovine chicken goat guinea pig syrian hamster horse human rabbit and sheep serum proteins but it may cross react with immunoglobulins from other species
    https://www.bioz.com/result/alexa fluor 647 conjugated anti rabbit/product/Jackson Immuno
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    alexa fluor 647 conjugated anti rabbit - by Bioz Stars, 2021-09
    98/100 stars

    Images

    1) Product Images from "Triciribine Engages ZFP36L1 and HuR to Stabilize LDLR mRNA"

    Article Title: Triciribine Engages ZFP36L1 and HuR to Stabilize LDLR mRNA

    Journal: Molecules

    doi: 10.3390/molecules25194505

    TCN induces translocation of nuclear HuR to cytoplasm in an ERK-dependent manner. ( a ) HepG2 cells were treated with, or without U0126 for one hour prior to addition of TCN or not for four hours and total lysate were immunoblotted with the indicated antibodies. ( b ) The cells were treated with TCN for four hours and subjected to subcellular fractionation with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents. Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. ( c ) Cells were treated with, or without, U0126 for one hour prior to the addition of TCN for four hours and subjected to subcellular fractionation and the cytosolic fraction was immunoblotted with the indicated antibodies. ( d ) HA-HuR transfected cells were exposed to TCN or PMA for four hours and further examined by confocal immunofluorescence microscopy with anti-HA. ( e ) Merged picture of HA-HuR and nuclei (Hoechst) in cells treated as described in ( d ) and with an increased resolution compared to ( d ). Red: HA-HuR, Alexa fluor 647, blue: nuclei, Hoechst. One representative experiment is shown ( n = 3). ( f ) Nuclear and cytoplasmic HA-HuR was localized in 600–900 cells for each treatment. The proportion of cells with nuclear and cytoplasmic HA-HuR, respectively, to the total number of cells counted, are shown ( n = 1).
    Figure Legend Snippet: TCN induces translocation of nuclear HuR to cytoplasm in an ERK-dependent manner. ( a ) HepG2 cells were treated with, or without U0126 for one hour prior to addition of TCN or not for four hours and total lysate were immunoblotted with the indicated antibodies. ( b ) The cells were treated with TCN for four hours and subjected to subcellular fractionation with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents. Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. ( c ) Cells were treated with, or without, U0126 for one hour prior to the addition of TCN for four hours and subjected to subcellular fractionation and the cytosolic fraction was immunoblotted with the indicated antibodies. ( d ) HA-HuR transfected cells were exposed to TCN or PMA for four hours and further examined by confocal immunofluorescence microscopy with anti-HA. ( e ) Merged picture of HA-HuR and nuclei (Hoechst) in cells treated as described in ( d ) and with an increased resolution compared to ( d ). Red: HA-HuR, Alexa fluor 647, blue: nuclei, Hoechst. One representative experiment is shown ( n = 3). ( f ) Nuclear and cytoplasmic HA-HuR was localized in 600–900 cells for each treatment. The proportion of cells with nuclear and cytoplasmic HA-HuR, respectively, to the total number of cells counted, are shown ( n = 1).

    Techniques Used: Translocation Assay, Fractionation, Transfection, Immunofluorescence, Microscopy

    2) Product Images from "Triciribine Engages ZFP36L1 and HuR to Stabilize LDLR mRNA"

    Article Title: Triciribine Engages ZFP36L1 and HuR to Stabilize LDLR mRNA

    Journal: Molecules

    doi: 10.3390/molecules25194505

    TCN induces translocation of nuclear HuR to cytoplasm in an ERK-dependent manner. ( a ) HepG2 cells were treated with, or without U0126 for one hour prior to addition of TCN or not for four hours and total lysate were immunoblotted with the indicated antibodies. ( b ) The cells were treated with TCN for four hours and subjected to subcellular fractionation with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents. Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. ( c ) Cells were treated with, or without, U0126 for one hour prior to the addition of TCN for four hours and subjected to subcellular fractionation and the cytosolic fraction was immunoblotted with the indicated antibodies. ( d ) HA-HuR transfected cells were exposed to TCN or PMA for four hours and further examined by confocal immunofluorescence microscopy with anti-HA. ( e ) Merged picture of HA-HuR and nuclei (Hoechst) in cells treated as described in ( d ) and with an increased resolution compared to ( d ). Red: HA-HuR, Alexa fluor 647, blue: nuclei, Hoechst. One representative experiment is shown ( n = 3). ( f ) Nuclear and cytoplasmic HA-HuR was localized in 600–900 cells for each treatment. The proportion of cells with nuclear and cytoplasmic HA-HuR, respectively, to the total number of cells counted, are shown ( n = 1).
    Figure Legend Snippet: TCN induces translocation of nuclear HuR to cytoplasm in an ERK-dependent manner. ( a ) HepG2 cells were treated with, or without U0126 for one hour prior to addition of TCN or not for four hours and total lysate were immunoblotted with the indicated antibodies. ( b ) The cells were treated with TCN for four hours and subjected to subcellular fractionation with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents. Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. ( c ) Cells were treated with, or without, U0126 for one hour prior to the addition of TCN for four hours and subjected to subcellular fractionation and the cytosolic fraction was immunoblotted with the indicated antibodies. ( d ) HA-HuR transfected cells were exposed to TCN or PMA for four hours and further examined by confocal immunofluorescence microscopy with anti-HA. ( e ) Merged picture of HA-HuR and nuclei (Hoechst) in cells treated as described in ( d ) and with an increased resolution compared to ( d ). Red: HA-HuR, Alexa fluor 647, blue: nuclei, Hoechst. One representative experiment is shown ( n = 3). ( f ) Nuclear and cytoplasmic HA-HuR was localized in 600–900 cells for each treatment. The proportion of cells with nuclear and cytoplasmic HA-HuR, respectively, to the total number of cells counted, are shown ( n = 1).

    Techniques Used: Translocation Assay, Fractionation, Transfection, Immunofluorescence, Microscopy

    Related Articles

    Binding Assay:

    Article Title: Genetic inactivation of SARM1 axon degeneration pathway improves outcome trajectory after experimental traumatic brain injury based on pathological, radiological, and functional measures
    Article Snippet: .. Microglia/macrophages were identified using polyclonal rabbit antibody against ionized calcium binding adaptor molecule 1 (IBA1; 1:500; Wako, Richmond, VA; Cat# 019-19741, RRID:AB_839504) followed by incubation with AlexaFluor-488-conjugated secondary antibody (1:400, Jackson ImmunoResearch Cat# 715-546-151, RRID: AB_2340850). ..

    Incubation:

    Article Title: Genetic inactivation of SARM1 axon degeneration pathway improves outcome trajectory after experimental traumatic brain injury based on pathological, radiological, and functional measures
    Article Snippet: .. Microglia/macrophages were identified using polyclonal rabbit antibody against ionized calcium binding adaptor molecule 1 (IBA1; 1:500; Wako, Richmond, VA; Cat# 019-19741, RRID:AB_839504) followed by incubation with AlexaFluor-488-conjugated secondary antibody (1:400, Jackson ImmunoResearch Cat# 715-546-151, RRID: AB_2340850). ..

    Article Title: Spermatogonia Loss Correlates with LAMA 1 Expression in Human Prepubertal Testes Stored for Fertility Preservation
    Article Snippet: .. After washing the slides in a TBS buffer, the secondary antibody incubation was performed at room temperature for 1 h in a 1:2 TBS diluted blocking buffer with a secondary antibody: Donkey anti rabbit Cy3 (711-166-152, Jackson Immuno Research) and donkey anti mouse AF488 (715-546-150, Jackson Immuno Research). ..

    Staining:

    Article Title: Sirt6 Regulates the Development of Medullary Thymic Epithelial Cells and Contributes to the Establishment of Central Immune Tolerance
    Article Snippet: .. The secondary antibodies were used for staining: Alexa Fluor 594-conjugated donkey anti-rabbit IgG (H + L) (Jackson ImmunoResearch Laboratories, 711-586-152) diluted by 1:400, Alexa Fluor 488-conjugated donkey anti-rat IgG (H + L) (Jackson ImmunoResearch Laboratories, 712-546-150) diluted by 1:400, and Alexa Fluor 488-conjugated donkey anti-mice IgG (H + L) antibodies (Jackson ImmunoResearch Laboratories, 715-546-150) diluted by 1:300. ..

    Blocking Assay:

    Article Title: Spermatogonia Loss Correlates with LAMA 1 Expression in Human Prepubertal Testes Stored for Fertility Preservation
    Article Snippet: .. After washing the slides in a TBS buffer, the secondary antibody incubation was performed at room temperature for 1 h in a 1:2 TBS diluted blocking buffer with a secondary antibody: Donkey anti rabbit Cy3 (711-166-152, Jackson Immuno Research) and donkey anti mouse AF488 (715-546-150, Jackson Immuno Research). ..

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  • 98
    Jackson Immuno alexa fluor 647 conjugated anti rabbit
    TCN induces translocation of nuclear HuR to cytoplasm in an ERK-dependent manner. ( a ) HepG2 cells were treated with, or without U0126 for one hour prior to addition of TCN or not for four hours and total lysate were immunoblotted with the indicated antibodies. ( b ) The cells were treated with TCN for four hours and subjected to subcellular fractionation with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents. Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. ( c ) Cells were treated with, or without, U0126 for one hour prior to the addition of TCN for four hours and subjected to subcellular fractionation and the cytosolic fraction was immunoblotted with the indicated antibodies. ( d ) HA-HuR transfected cells were exposed to TCN or PMA for four hours and further examined by confocal immunofluorescence microscopy with anti-HA. ( e ) Merged picture of HA-HuR and nuclei (Hoechst) in cells treated as described in ( d ) and with an increased resolution compared to ( d ). Red: HA-HuR, <t>Alexa</t> fluor 647, blue: nuclei, Hoechst. One representative experiment is shown ( n = 3). ( f ) Nuclear and cytoplasmic HA-HuR was localized in 600–900 cells for each treatment. The proportion of cells with nuclear and cytoplasmic HA-HuR, respectively, to the total number of cells counted, are shown ( n = 1).
    Alexa Fluor 647 Conjugated Anti Rabbit, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 647 conjugated anti rabbit/product/Jackson Immuno
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    alexa fluor 647 conjugated anti rabbit - by Bioz Stars, 2021-09
    98/100 stars
      Buy from Supplier

    93
    Jackson Immuno alexafluor 647 conjugated rabbit anti dog igg
    Establishment of chimeric and caninized anti-PD-1 monoclonal antibodies. ( A ) Schematic overview of rat monoclonal antibody (mAb) 4F12-E6, rat-dog chimeric mAb ch-4F12-E6, and caninized mAb ca-4F12-E6. ( B ) NRK cells expressing canine PD-1 (NRK/cPD-1) were stained with 40 μg/ml of rat IgG2a isotype control, 4F12-E6, ch-4F12-E6, or ca-4F12-E6 followed by incubation with secondary antibodies, anti-rat IgG-Dylight 649, or anti-dog IgG-AlexaFluor 647. As a control, cells were stained with anti-dog IgG-AlexaFluor 647 without primary antibody (indicated as Secondary Ab only). No antibodies bound to NRK cells transfected with empty vector. ( C ) NRK/cPD-1 cells were pre-incubated with dose-titrated monoclonal antibodies for ch-4F12-E6 or ca-4F12-E6, and, after washing, incubated with 40 μg/ml of cPD-L1-Ig or human Ig. Cells were then stained with secondary antibody anti-human IgG-PE. Representative data from at least two biological replicates are shown.
    Alexafluor 647 Conjugated Rabbit Anti Dog Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexafluor 647 conjugated rabbit anti dog igg/product/Jackson Immuno
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    alexafluor 647 conjugated rabbit anti dog igg - by Bioz Stars, 2021-09
    93/100 stars
      Buy from Supplier

    99
    Jackson Immuno anti rabbit igg
    Endogenous ATG16L1 + or WIPI2 + phagophores do not colocalize with recycling endosomes. (A) A549 cells were starved with EBSS for 2 h. Cells were then fixed and co-probed with ATG16L1 antibody and TFRC antibody. <t>Alexa</t> Fluor 488-conjugated goat anti-rabbit <t>IgG-</t> and Alexa Fluor 647-conjugated donkey anti-mouse IgG secondary antibodies were used to detect ATG16L1 and TFRC + RE, respectively. Boxed area 1 and 2 are enlarged to show the colocalization between ATG16 + puncta and TFRC-labeled recycling endosomes. (B) A549 cells were starved with EBSS for 2 h. Cells were then fixed and co-probed with RAB11 antibody and WIPI2 antibody. Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 647-conjugated donkey anti-mouse IgG secondary antibodies were used to detect RAB11 + REs and WIPI2 + phagophores, respectively. (C) A549 cells were starved with EBSS plus fluorescein-conjugated human TRF for 2 h. Cells were then fixed and probed with WIPI2 antibody. Alexa Fluor 647-conjugated donkey anti-mouse IgG secondary antibody was used to detect WIPI2 + phagophores. Boxed area is enlarged to show the details. Scale bar: 25 micron.
    Anti Rabbit Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rabbit igg/product/Jackson Immuno
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti rabbit igg - by Bioz Stars, 2021-09
    99/100 stars
      Buy from Supplier

    99
    Jackson Immuno alexa 647 conjugated donkey anti rabbit igg
    Gephyrin SSDs and their alignment with pre‐synaptic RIM1/2  in vivo dSTORM imaging of gephyrin in sucrose impregnated cryosections of adult mouse spinal cord. (A) dSTORM detections of gephyrin (red dots) overlaid with the epifluorescence image (white with gray background). Synaptic clusters of gephyrin in dSTORM images were identified by the epifluorescence puncta. Scale bar: 1 µm. (B) Enlarged pointillist images of the two gephyrin clusters indicated in (A). (C) Rendered images of the two gephyrin clusters outlined with the boundaries of the epifluorescence mask. Gephyrin SSDs are indicated with red asterisks in (B) and (C). Scale bar: 200 nm. Two‐color dSTORM imaging of RIM1/2 and gephyrin in cryosections. From left to right: rendered images of RIM1/2 and gephyrin clusters showing the aligned SSDs (arrows); gephyrin SSDs segmented by H‐watershed outlined with different colors; RIM1/2 SSDs outlined with different colors; binary SSDs of gephyrin and RIM1/2. Inhibitory synapses were identified by the gephyrin clusters in the epifluorescence images. Scale bar: 200 nm. RIM1/2 was labeled with Alexa 647, gephyrin (mAb7a) with Cy3B in these experiments. Source data are available online for this figure.
    Alexa 647 Conjugated Donkey Anti Rabbit Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa 647 conjugated donkey anti rabbit igg/product/Jackson Immuno
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    alexa 647 conjugated donkey anti rabbit igg - by Bioz Stars, 2021-09
    99/100 stars
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    Image Search Results


    TCN induces translocation of nuclear HuR to cytoplasm in an ERK-dependent manner. ( a ) HepG2 cells were treated with, or without U0126 for one hour prior to addition of TCN or not for four hours and total lysate were immunoblotted with the indicated antibodies. ( b ) The cells were treated with TCN for four hours and subjected to subcellular fractionation with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents. Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. ( c ) Cells were treated with, or without, U0126 for one hour prior to the addition of TCN for four hours and subjected to subcellular fractionation and the cytosolic fraction was immunoblotted with the indicated antibodies. ( d ) HA-HuR transfected cells were exposed to TCN or PMA for four hours and further examined by confocal immunofluorescence microscopy with anti-HA. ( e ) Merged picture of HA-HuR and nuclei (Hoechst) in cells treated as described in ( d ) and with an increased resolution compared to ( d ). Red: HA-HuR, Alexa fluor 647, blue: nuclei, Hoechst. One representative experiment is shown ( n = 3). ( f ) Nuclear and cytoplasmic HA-HuR was localized in 600–900 cells for each treatment. The proportion of cells with nuclear and cytoplasmic HA-HuR, respectively, to the total number of cells counted, are shown ( n = 1).

    Journal: Molecules

    Article Title: Triciribine Engages ZFP36L1 and HuR to Stabilize LDLR mRNA

    doi: 10.3390/molecules25194505

    Figure Lengend Snippet: TCN induces translocation of nuclear HuR to cytoplasm in an ERK-dependent manner. ( a ) HepG2 cells were treated with, or without U0126 for one hour prior to addition of TCN or not for four hours and total lysate were immunoblotted with the indicated antibodies. ( b ) The cells were treated with TCN for four hours and subjected to subcellular fractionation with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents. Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. ( c ) Cells were treated with, or without, U0126 for one hour prior to the addition of TCN for four hours and subjected to subcellular fractionation and the cytosolic fraction was immunoblotted with the indicated antibodies. ( d ) HA-HuR transfected cells were exposed to TCN or PMA for four hours and further examined by confocal immunofluorescence microscopy with anti-HA. ( e ) Merged picture of HA-HuR and nuclei (Hoechst) in cells treated as described in ( d ) and with an increased resolution compared to ( d ). Red: HA-HuR, Alexa fluor 647, blue: nuclei, Hoechst. One representative experiment is shown ( n = 3). ( f ) Nuclear and cytoplasmic HA-HuR was localized in 600–900 cells for each treatment. The proportion of cells with nuclear and cytoplasmic HA-HuR, respectively, to the total number of cells counted, are shown ( n = 1).

    Article Snippet: Alexa Fluor 647-conjugated anti-rabbit (1:200 dilution; 715–546–150), (Jackson Immuno Research, Cambridge, UK) was used for visualization.

    Techniques: Translocation Assay, Fractionation, Transfection, Immunofluorescence, Microscopy

    Establishment of chimeric and caninized anti-PD-1 monoclonal antibodies. ( A ) Schematic overview of rat monoclonal antibody (mAb) 4F12-E6, rat-dog chimeric mAb ch-4F12-E6, and caninized mAb ca-4F12-E6. ( B ) NRK cells expressing canine PD-1 (NRK/cPD-1) were stained with 40 μg/ml of rat IgG2a isotype control, 4F12-E6, ch-4F12-E6, or ca-4F12-E6 followed by incubation with secondary antibodies, anti-rat IgG-Dylight 649, or anti-dog IgG-AlexaFluor 647. As a control, cells were stained with anti-dog IgG-AlexaFluor 647 without primary antibody (indicated as Secondary Ab only). No antibodies bound to NRK cells transfected with empty vector. ( C ) NRK/cPD-1 cells were pre-incubated with dose-titrated monoclonal antibodies for ch-4F12-E6 or ca-4F12-E6, and, after washing, incubated with 40 μg/ml of cPD-L1-Ig or human Ig. Cells were then stained with secondary antibody anti-human IgG-PE. Representative data from at least two biological replicates are shown.

    Journal: Scientific Reports

    Article Title: A pilot clinical study of the therapeutic antibody against canine PD-1 for advanced spontaneous cancers in dogs

    doi: 10.1038/s41598-020-75533-4

    Figure Lengend Snippet: Establishment of chimeric and caninized anti-PD-1 monoclonal antibodies. ( A ) Schematic overview of rat monoclonal antibody (mAb) 4F12-E6, rat-dog chimeric mAb ch-4F12-E6, and caninized mAb ca-4F12-E6. ( B ) NRK cells expressing canine PD-1 (NRK/cPD-1) were stained with 40 μg/ml of rat IgG2a isotype control, 4F12-E6, ch-4F12-E6, or ca-4F12-E6 followed by incubation with secondary antibodies, anti-rat IgG-Dylight 649, or anti-dog IgG-AlexaFluor 647. As a control, cells were stained with anti-dog IgG-AlexaFluor 647 without primary antibody (indicated as Secondary Ab only). No antibodies bound to NRK cells transfected with empty vector. ( C ) NRK/cPD-1 cells were pre-incubated with dose-titrated monoclonal antibodies for ch-4F12-E6 or ca-4F12-E6, and, after washing, incubated with 40 μg/ml of cPD-L1-Ig or human Ig. Cells were then stained with secondary antibody anti-human IgG-PE. Representative data from at least two biological replicates are shown.

    Article Snippet: Harvested cells were stained with primary antibodies (40 μg/ml) including rat IgG2a isotype control (BioLegend, San Diego, CA, USA), 4F12-E6, ch-4F12-E6, and ca-4F12-E6, followed by incubation with Dylight 649-conjugated goat anti-rat IgG (BioLegend) or AlexaFluor 647-conjugated rabbit anti-dog IgG (Jackson ImmunoResearch, West Grove, PA, USA).

    Techniques: Expressing, Staining, Incubation, Transfection, Plasmid Preparation

    Endogenous ATG16L1 + or WIPI2 + phagophores do not colocalize with recycling endosomes. (A) A549 cells were starved with EBSS for 2 h. Cells were then fixed and co-probed with ATG16L1 antibody and TFRC antibody. Alexa Fluor 488-conjugated goat anti-rabbit IgG- and Alexa Fluor 647-conjugated donkey anti-mouse IgG secondary antibodies were used to detect ATG16L1 and TFRC + RE, respectively. Boxed area 1 and 2 are enlarged to show the colocalization between ATG16 + puncta and TFRC-labeled recycling endosomes. (B) A549 cells were starved with EBSS for 2 h. Cells were then fixed and co-probed with RAB11 antibody and WIPI2 antibody. Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 647-conjugated donkey anti-mouse IgG secondary antibodies were used to detect RAB11 + REs and WIPI2 + phagophores, respectively. (C) A549 cells were starved with EBSS plus fluorescein-conjugated human TRF for 2 h. Cells were then fixed and probed with WIPI2 antibody. Alexa Fluor 647-conjugated donkey anti-mouse IgG secondary antibody was used to detect WIPI2 + phagophores. Boxed area is enlarged to show the details. Scale bar: 25 micron.

    Journal: Autophagy

    Article Title: Transiently expressed ATG16L1 inhibits autophagosome biogenesis and aberrantly targets RAB11-positive recycling endosomes

    doi: 10.1080/15548627.2016.1256521

    Figure Lengend Snippet: Endogenous ATG16L1 + or WIPI2 + phagophores do not colocalize with recycling endosomes. (A) A549 cells were starved with EBSS for 2 h. Cells were then fixed and co-probed with ATG16L1 antibody and TFRC antibody. Alexa Fluor 488-conjugated goat anti-rabbit IgG- and Alexa Fluor 647-conjugated donkey anti-mouse IgG secondary antibodies were used to detect ATG16L1 and TFRC + RE, respectively. Boxed area 1 and 2 are enlarged to show the colocalization between ATG16 + puncta and TFRC-labeled recycling endosomes. (B) A549 cells were starved with EBSS for 2 h. Cells were then fixed and co-probed with RAB11 antibody and WIPI2 antibody. Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 647-conjugated donkey anti-mouse IgG secondary antibodies were used to detect RAB11 + REs and WIPI2 + phagophores, respectively. (C) A549 cells were starved with EBSS plus fluorescein-conjugated human TRF for 2 h. Cells were then fixed and probed with WIPI2 antibody. Alexa Fluor 647-conjugated donkey anti-mouse IgG secondary antibody was used to detect WIPI2 + phagophores. Boxed area is enlarged to show the details. Scale bar: 25 micron.

    Article Snippet: RAB11 antibody (ThermoFisher Scientific, 71–5300); TFRC antibody (ThermoFisher Scientific, 236–15375); EEA1 antibody (BD Biosciences, 610457); GOLGA2/GM130 antibody (BD Biosciences, 610822); ATG16L1 antibody (Cell Signaling Technology, 8089); WIPI2 antibody (Bio-Rad, MCA5780GA); MAP1LC3B antibody (Cell Signaling Technology, 3868); ATG7 antibody (Cell Signaling Technology, 8558); anti-human ATG12 (Cell Signaling Technology, 2010) and anti-mouse ATG12 (Cell Signaling Technology, 2011); RAB7 antibody (Cell Signaling Technology, 9367); SQSTM1 antibody (Santa Cruz Biotechnology, sc-28359); ULK1 antibody (H-240; Santa Cruz Biotechnology, 33182); LAMP2 antibody (Santa Cruz Biotechnology, sc-18822); LMAN1/ERGIC-53 antibody (C-6; Santa Cruz Biotechnology, sc-365158); anti ACTB/β-actin (Santa Cruz Biotechnology, sc-47778); anti-rabbit IgG (Alexa Fluor 488 or 555 conjugate) (Cell Signaling Technology, 4412; 4413); anti-mouse IgG (Alexa Fluor 488 or 555 conjugate) (Cell Signaling Technology, 4408; 4409); donkey anti-mouse or anti-rabbit IgG (Alexa Fluor 647 conjugate; Jackson ImmunoResearch Laboratories, 715–605–150; 711–175–152).

    Techniques: Labeling

    Gephyrin SSDs and their alignment with pre‐synaptic RIM1/2  in vivo dSTORM imaging of gephyrin in sucrose impregnated cryosections of adult mouse spinal cord. (A) dSTORM detections of gephyrin (red dots) overlaid with the epifluorescence image (white with gray background). Synaptic clusters of gephyrin in dSTORM images were identified by the epifluorescence puncta. Scale bar: 1 µm. (B) Enlarged pointillist images of the two gephyrin clusters indicated in (A). (C) Rendered images of the two gephyrin clusters outlined with the boundaries of the epifluorescence mask. Gephyrin SSDs are indicated with red asterisks in (B) and (C). Scale bar: 200 nm. Two‐color dSTORM imaging of RIM1/2 and gephyrin in cryosections. From left to right: rendered images of RIM1/2 and gephyrin clusters showing the aligned SSDs (arrows); gephyrin SSDs segmented by H‐watershed outlined with different colors; RIM1/2 SSDs outlined with different colors; binary SSDs of gephyrin and RIM1/2. Inhibitory synapses were identified by the gephyrin clusters in the epifluorescence images. Scale bar: 200 nm. RIM1/2 was labeled with Alexa 647, gephyrin (mAb7a) with Cy3B in these experiments. Source data are available online for this figure.

    Journal: EMBO Reports

    Article Title: Differential regulation of glycinergic and GABAergic nanocolumns at mixed inhibitory synapses

    doi: 10.15252/embr.202052154

    Figure Lengend Snippet: Gephyrin SSDs and their alignment with pre‐synaptic RIM1/2 in vivo dSTORM imaging of gephyrin in sucrose impregnated cryosections of adult mouse spinal cord. (A) dSTORM detections of gephyrin (red dots) overlaid with the epifluorescence image (white with gray background). Synaptic clusters of gephyrin in dSTORM images were identified by the epifluorescence puncta. Scale bar: 1 µm. (B) Enlarged pointillist images of the two gephyrin clusters indicated in (A). (C) Rendered images of the two gephyrin clusters outlined with the boundaries of the epifluorescence mask. Gephyrin SSDs are indicated with red asterisks in (B) and (C). Scale bar: 200 nm. Two‐color dSTORM imaging of RIM1/2 and gephyrin in cryosections. From left to right: rendered images of RIM1/2 and gephyrin clusters showing the aligned SSDs (arrows); gephyrin SSDs segmented by H‐watershed outlined with different colors; RIM1/2 SSDs outlined with different colors; binary SSDs of gephyrin and RIM1/2. Inhibitory synapses were identified by the gephyrin clusters in the epifluorescence images. Scale bar: 200 nm. RIM1/2 was labeled with Alexa 647, gephyrin (mAb7a) with Cy3B in these experiments. Source data are available online for this figure.

    Article Snippet: Sections were then incubated with Alexa 647‐conjugated donkey anti‐rabbit IgG (Jackson ImmunoResearch, Cat. No. 711‐605‐152, dilution 1:500) and laboratory‐made Cy3B‐conjugated donkey anti‐mouse secondary antibodies (dilution 1:100) for 2 h at room temperature.

    Techniques: In Vivo, Imaging, Labeling

    Trans‐synaptic alignment at inhibitory synapses in cultured spinal cord neurons Alignment of SSDs of pre‐synaptic RIM1/2 and SSDs of post‐synaptic gephyrin, GlyRs, and GABA A Rs, respectively. (A1–C1) Distances were measured between the intensity peaks of paired SSDs in rendered dual‐color dSTORM images (yellow line, values given in mean ± SD). RIM1/2 was labeled with Alexa 647 and gephyrin (mAb7a) with Cy3B in (A1). RIM1/2 was labeled with Cy3B, while GlyRs or GABAARs were labeled with Alexa 647 in (B1) and (C1). (A2–C2) Trans‐synaptic alignment of SSDs of gephyrin, GlyRs, GABA A Rs, and RIM1/2. SSDs were segmented by H‐watershed. Data are plotted as mean ± SD. Number of synapses: n = 48 (A1, A2) from three independent experiments (no treatment), n = 30 (B1, B2) from two experiments with TTX treatment, n = 16 (C1, C2) from two experiments with TTX. Only synapses with side view profiles were included. All images are adjusted to the same scale. Scale bar: 200 nm. Source data are available online for this figure.

    Journal: EMBO Reports

    Article Title: Differential regulation of glycinergic and GABAergic nanocolumns at mixed inhibitory synapses

    doi: 10.15252/embr.202052154

    Figure Lengend Snippet: Trans‐synaptic alignment at inhibitory synapses in cultured spinal cord neurons Alignment of SSDs of pre‐synaptic RIM1/2 and SSDs of post‐synaptic gephyrin, GlyRs, and GABA A Rs, respectively. (A1–C1) Distances were measured between the intensity peaks of paired SSDs in rendered dual‐color dSTORM images (yellow line, values given in mean ± SD). RIM1/2 was labeled with Alexa 647 and gephyrin (mAb7a) with Cy3B in (A1). RIM1/2 was labeled with Cy3B, while GlyRs or GABAARs were labeled with Alexa 647 in (B1) and (C1). (A2–C2) Trans‐synaptic alignment of SSDs of gephyrin, GlyRs, GABA A Rs, and RIM1/2. SSDs were segmented by H‐watershed. Data are plotted as mean ± SD. Number of synapses: n = 48 (A1, A2) from three independent experiments (no treatment), n = 30 (B1, B2) from two experiments with TTX treatment, n = 16 (C1, C2) from two experiments with TTX. Only synapses with side view profiles were included. All images are adjusted to the same scale. Scale bar: 200 nm. Source data are available online for this figure.

    Article Snippet: Sections were then incubated with Alexa 647‐conjugated donkey anti‐rabbit IgG (Jackson ImmunoResearch, Cat. No. 711‐605‐152, dilution 1:500) and laboratory‐made Cy3B‐conjugated donkey anti‐mouse secondary antibodies (dilution 1:100) for 2 h at room temperature.

    Techniques: Cell Culture, Labeling