dab substrate kit  (Thermo Fisher)


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    Pierce DAB Substrate Kit
    Description:
    The Thermo Scientific Pierce DAB Substrate Kit enables chromogenic detection of horseradish peroxidase HRP activity based the action of 3 3 diaminobenzidine DAB in Western blot and tissue staining methods DAB 3 3 diaminobenzidine tetrahydrochloride MW 214 1 reacts with HRP in the presence of peroxide to yield an insoluble brown colored product at locations where peroxidase conjugated antibodies are bound to samples The brown precipitate is insoluble in alcohol and other organic solvents making it an excellent substrate for immunohistochemical staining that requires the use of traditional counterstains and mounting media The Pierce DAB Substrate Kit includes a 10X solution of DAB and Stable Peroxide Substrate Buffer Crystalline DAB powder is also available Features DAB Substrate • HRP substrate for detection of horseradish peroxidase activity on solid media including nitrocellulose and PVDF membranes and fixed tissue samples • Chromogenic no special equipment needed for visualization the DAB reaction produces brown bands or spots at sites of reaction with HRP conjugated antibodies or probes • Package options choose powder for custom formulation with buffer and peroxide or a complete kit of preformulated solutions for immediate use Features of the Pierce DAB Substrate Kit • Easy to use dilute 10X stock solution to 1X in provided stable peroxide buffer • Safer preformulated DAB solution minimizes safety concerns with this chemical • Complete includes convenient solution of stable hydrogen peroxide buffer Part No 34062 • Stable store refrigerated at 4°C for at least one year freezer storage is not necessary DAB has been used in a variety of applications including Western blotting immunohistochemistry and electron microscopy Many enhancement methods have been reported for DAB involving different buffer conditions the addition of metal ions and post treatment applications Ref 1 7 also see the Pierce Metal Enhanced DAB Substrate Kit Part No 34065 One disadvantage of DAB compared to other substrates is the need to use the working solution immediately because it begins a reaction process upon the addition of hydrogen peroxide This results in increased background color of the substrate However the brown bands resulting from DAB reactions do not fade as quickly as the color products of other precipitating HRP substrates Related Products DAB Substrate Powder 3 3 diaminobenzidine tetrahydrochloride
    Catalog Number:
    34002
    Price:
    None
    Category:
    Labeling Detection Products
    Applications:
    Cell Analysis|Cellular Imaging|IHC Staining & Detection|Immunohistochemistry (IHC)|Protein Assays and Analysis|Protein Biology|Western Blotting
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    Structured Review

    Thermo Fisher dab substrate kit
    Enzyme-histochemical images of <t>DAB</t> reaction of <t>HRP</t> in cryosections of mouse thymic tissues, which were injected with various concentrations of HRP [10 ( c, d ), 25 ( e, f ), 50 ( g, h ) and 100 mg/ml ( i, j )] via left ventricles, and prepared at 3 min after HRP injection by IVCT. ( a, b ) Control mice (Con) without HRP injection. MG, methyl green staining. ( c–j ) HRP-enzyme reactivities are variously detected in the interstitium (arrows) around some blood vessels in corticomedullary boundary areas and other thick blood vessels of the medulla, depending on the HRP concentration, but they are also found within blood vessels (arrowheads) in addition to tiny blood capillaries. Co, cortex. M, medulla. Bars=20 μm.
    The Thermo Scientific Pierce DAB Substrate Kit enables chromogenic detection of horseradish peroxidase HRP activity based the action of 3 3 diaminobenzidine DAB in Western blot and tissue staining methods DAB 3 3 diaminobenzidine tetrahydrochloride MW 214 1 reacts with HRP in the presence of peroxide to yield an insoluble brown colored product at locations where peroxidase conjugated antibodies are bound to samples The brown precipitate is insoluble in alcohol and other organic solvents making it an excellent substrate for immunohistochemical staining that requires the use of traditional counterstains and mounting media The Pierce DAB Substrate Kit includes a 10X solution of DAB and Stable Peroxide Substrate Buffer Crystalline DAB powder is also available Features DAB Substrate • HRP substrate for detection of horseradish peroxidase activity on solid media including nitrocellulose and PVDF membranes and fixed tissue samples • Chromogenic no special equipment needed for visualization the DAB reaction produces brown bands or spots at sites of reaction with HRP conjugated antibodies or probes • Package options choose powder for custom formulation with buffer and peroxide or a complete kit of preformulated solutions for immediate use Features of the Pierce DAB Substrate Kit • Easy to use dilute 10X stock solution to 1X in provided stable peroxide buffer • Safer preformulated DAB solution minimizes safety concerns with this chemical • Complete includes convenient solution of stable hydrogen peroxide buffer Part No 34062 • Stable store refrigerated at 4°C for at least one year freezer storage is not necessary DAB has been used in a variety of applications including Western blotting immunohistochemistry and electron microscopy Many enhancement methods have been reported for DAB involving different buffer conditions the addition of metal ions and post treatment applications Ref 1 7 also see the Pierce Metal Enhanced DAB Substrate Kit Part No 34065 One disadvantage of DAB compared to other substrates is the need to use the working solution immediately because it begins a reaction process upon the addition of hydrogen peroxide This results in increased background color of the substrate However the brown bands resulting from DAB reactions do not fade as quickly as the color products of other precipitating HRP substrates Related Products DAB Substrate Powder 3 3 diaminobenzidine tetrahydrochloride
    https://www.bioz.com/result/dab substrate kit/product/Thermo Fisher
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dab substrate kit - by Bioz Stars, 2021-05
    95/100 stars

    Images

    1) Product Images from "Immuno- and Enzyme-histochemistry of HRP for Demonstration of Blood Vessel Permeability in Mouse Thymic Tissues by “In Vivo Cryotechnique”"

    Article Title: Immuno- and Enzyme-histochemistry of HRP for Demonstration of Blood Vessel Permeability in Mouse Thymic Tissues by “In Vivo Cryotechnique”

    Journal: Acta Histochemica et Cytochemica

    doi: 10.1267/ahc.14038

    Enzyme-histochemical images of DAB reaction of HRP in cryosections of mouse thymic tissues, which were injected with various concentrations of HRP [10 ( c, d ), 25 ( e, f ), 50 ( g, h ) and 100 mg/ml ( i, j )] via left ventricles, and prepared at 3 min after HRP injection by IVCT. ( a, b ) Control mice (Con) without HRP injection. MG, methyl green staining. ( c–j ) HRP-enzyme reactivities are variously detected in the interstitium (arrows) around some blood vessels in corticomedullary boundary areas and other thick blood vessels of the medulla, depending on the HRP concentration, but they are also found within blood vessels (arrowheads) in addition to tiny blood capillaries. Co, cortex. M, medulla. Bars=20 μm.
    Figure Legend Snippet: Enzyme-histochemical images of DAB reaction of HRP in cryosections of mouse thymic tissues, which were injected with various concentrations of HRP [10 ( c, d ), 25 ( e, f ), 50 ( g, h ) and 100 mg/ml ( i, j )] via left ventricles, and prepared at 3 min after HRP injection by IVCT. ( a, b ) Control mice (Con) without HRP injection. MG, methyl green staining. ( c–j ) HRP-enzyme reactivities are variously detected in the interstitium (arrows) around some blood vessels in corticomedullary boundary areas and other thick blood vessels of the medulla, depending on the HRP concentration, but they are also found within blood vessels (arrowheads) in addition to tiny blood capillaries. Co, cortex. M, medulla. Bars=20 μm.

    Techniques Used: Injection, Mouse Assay, Staining, Concentration Assay

    Enzyme-histochemical images of DAB reaction of HRP in cryosections of mouse thymic tissues, which were injected with HRP (100 mg/ml) via left ventricles, and prepared after various time intervals by IVCT. ( a, b ) Control mice (Con) without HRP injection. Arrowheads: blood vessels. MG, methyl green staining. ( c, d ) At 30 sec, HRP reaction products are slightly detected in the interstitium around some thick blood vessels in corticomedullary boundary areas (arrows) and also within other blood vessels (arrowheads). ( e–h ) At 1 min and 3 min, they are more widely detected in the interstitium around thick blood vessels in corticomedullary boundary areas (arrows) and within some thick blood vessels in both cortical and medullary areas (arrowheads). ( i, j ) At 10 min, they are diffusely detected in all the interstitium of cortical and medullary areas (arrows). ( k, l ) At 15 min, they are uniformly distributed throughout all the interstitium of thymic tissues (arrows) in addition to perivascular spaces of blood vessels (arrowheads). ( m, n ) At 30 min, HRP phagocytosis by macrophages (double arrowheads) is seen to be scattered, which is accompanied by the decrease of DAB reaction intensity in all the interstitial matrix. Arrowhead: blood vessel. Long arrow: nucleus. Short arrow: process. Co, cortex. M, medulla. Bars=20 μm.
    Figure Legend Snippet: Enzyme-histochemical images of DAB reaction of HRP in cryosections of mouse thymic tissues, which were injected with HRP (100 mg/ml) via left ventricles, and prepared after various time intervals by IVCT. ( a, b ) Control mice (Con) without HRP injection. Arrowheads: blood vessels. MG, methyl green staining. ( c, d ) At 30 sec, HRP reaction products are slightly detected in the interstitium around some thick blood vessels in corticomedullary boundary areas (arrows) and also within other blood vessels (arrowheads). ( e–h ) At 1 min and 3 min, they are more widely detected in the interstitium around thick blood vessels in corticomedullary boundary areas (arrows) and within some thick blood vessels in both cortical and medullary areas (arrowheads). ( i, j ) At 10 min, they are diffusely detected in all the interstitium of cortical and medullary areas (arrows). ( k, l ) At 15 min, they are uniformly distributed throughout all the interstitium of thymic tissues (arrows) in addition to perivascular spaces of blood vessels (arrowheads). ( m, n ) At 30 min, HRP phagocytosis by macrophages (double arrowheads) is seen to be scattered, which is accompanied by the decrease of DAB reaction intensity in all the interstitial matrix. Arrowhead: blood vessel. Long arrow: nucleus. Short arrow: process. Co, cortex. M, medulla. Bars=20 μm.

    Techniques Used: Injection, Mouse Assay, Staining, Size-exclusion Chromatography

    Related Articles

    Incubation:

    Article Title: The oppD Gene and Putative Peptidase Genes May Be Required for Virulence in Mycoplasma gallisepticum
    Article Snippet: Total cell proteins of mutants with protein profiles that differed in Coomassie-stained gels were separated in a 10% polyacrylamide gel along with molecular mass standards (PageRuler prestained protein ladder; Thermo Scientific) and electrophoretically transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P transfer membrane; Millipore), and antigen-free sites were blocked by overnight incubation in 5% skim milk (Devondale) in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T) at 4°C. .. The membrane was then washed three times (10 min each time) in PBS-T and then incubated in pooled sera collected from birds infected with Ap3AS (1:2,000 in PBS-T) at room temperature for 1 h with gentle rocking, again washed as described above, and then incubated for 1 h at room temperature in anti-chicken immunoglobulin-horseradish peroxidase conjugate (Dako) at a dilution of 1:2,000 in PBS-T. After again washing as described above, bound conjugate was detected using enhanced chemiluminescence (CD/DAB substrate kit; Thermo Scientific) following the manufacturer's recommendations, and the results were recorded digitally using a Bio-Rad molecular imager (Bio-Rad) according to the manufacturer's instructions. ..

    Article Title: Platelet-Derived Growth Factor Subunit B Signaling Promotes Pericyte Migration in Response to Loud Sound in the Cochlear Stria Vascularis
    Article Snippet: .. Cochleae were isolated and fixed with phosphate-buffered 3 % glutaraldehyde and 1.5 % PFA for 5 h. Stria vascularis from control and loud sound-stimulated groups were isolated and incubated in reaction buffer (Pierce™ DAB Substrate Kit, 34002, Thermo Fisher Scientific, USA) containing DAB and stable peroxide substrate for 1 h. After chemical reaction, samples were post-fixed for 1 h in 1 % osmium. .. Tissues were dehydrated through a graded alcohol series, embedded in Embed 812 (Electron Microscopy Sciences, Hatfield, PA., USA), sectioned, stained for 2 min with UranyLess (Electron Microscopy Sciences, USA), and examined on an FEI Tecnai-12 BioTWIN transmission electron microscope.

    Infection:

    Article Title: The oppD Gene and Putative Peptidase Genes May Be Required for Virulence in Mycoplasma gallisepticum
    Article Snippet: Total cell proteins of mutants with protein profiles that differed in Coomassie-stained gels were separated in a 10% polyacrylamide gel along with molecular mass standards (PageRuler prestained protein ladder; Thermo Scientific) and electrophoretically transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P transfer membrane; Millipore), and antigen-free sites were blocked by overnight incubation in 5% skim milk (Devondale) in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T) at 4°C. .. The membrane was then washed three times (10 min each time) in PBS-T and then incubated in pooled sera collected from birds infected with Ap3AS (1:2,000 in PBS-T) at room temperature for 1 h with gentle rocking, again washed as described above, and then incubated for 1 h at room temperature in anti-chicken immunoglobulin-horseradish peroxidase conjugate (Dako) at a dilution of 1:2,000 in PBS-T. After again washing as described above, bound conjugate was detected using enhanced chemiluminescence (CD/DAB substrate kit; Thermo Scientific) following the manufacturer's recommendations, and the results were recorded digitally using a Bio-Rad molecular imager (Bio-Rad) according to the manufacturer's instructions. ..

    Avidin-Biotin Assay:

    Article Title: Immuno- and Enzyme-histochemistry of HRP for Demonstration of Blood Vessel Permeability in Mouse Thymic Tissues by “In Vivo Cryotechnique”
    Article Snippet: The immunostained sections were additionally incubated with biotin-conjugated rabbit polyclonal anti-goat IgG antibody or biotin-conjugated goat polyclonal anti-rabbit IgG antibody (Vector, Burlingame, CA, USA) at room temperature for 1 hr. .. The immunoreaction products were visualized with the avidin–biotin–HRP complex (ABC) method (Vector) and a DAB substrate kit (Pierce, Rockford, IL, USA), and additionally fixed with 0.05% osmium tetroxide solution, as reported previously [ , ]. .. All immunostained sections were counterstained with methyl green, and finally observed with a light microscope (BX-61; Olympus, Tokyo, Japan) ( p).

    Article Title: Synergistic suppression effect on tumor growth of acute myeloid leukemia by combining cytarabine with an engineered oncolytic vaccinia virus
    Article Snippet: After 25 days, the mice were sacrificed, and the tumors were excised, treated with 4% paraformaldehyde, and encased in paraffin for hematoxylin & eosin (H & E) staining, immunohistochemistry, and TUNEL assay. .. For the immunohistochemical (IHC) study, the sections were stained with anti-Caspase-3 or anti-ING4 (1:100 dilutions) primary antibodies overnight at 4°C, added with the avidin-biotin-peroxidase complex, and the slides were detected with diaminobenzidine Kit (Thermo Fisher Scientific). .. Images were photographed by an inverted fluorescence microscope (Nikon E300; Tokyo, Japan).

    Article Title: A Combination of NT-4/5 and GDNF Is Favorable for Cultured Human Nigral Neural Progenitor Cells
    Article Snippet: After washing, sections were incubated with biotinylated anti-rabbit antibodies (1:200; Vector, Burlingame, CA, USA). .. Bound antibodies were visualized with the avidin–peroxidase complex method and the metal enhanced 3,3′-diaminobenzidine-based kit (Pierce, Rockford, IL, USA). .. Slides were then washed, dehydrated, and covered with Eukitt (O. Kindler GmbH, Freiburg, Germany).

    Article Title: Endothelial Progenitor Cell-Derived Factors Exert Neuroprotection in Cultured Cortical Neuronal Progenitor Cells
    Article Snippet: Endogenous peroxidase was blocked for 10 min in 3% hydrogen peroxide and 10% methanol in PBS. .. Bound antibodies were visualized after washes in PBS by the avidin–biotin complex (VECTASTAIN® ABC-Peroxidase Kit; 1:250, PK-4000; Vector Laboratories) in combination with the DAB Substrate Kit (34002, ThermoFischer Scientific). .. The cultures were mounted with Aquatex (Millipore, Darmstadt, Germany).

    Plasmid Preparation:

    Article Title: Immuno- and Enzyme-histochemistry of HRP for Demonstration of Blood Vessel Permeability in Mouse Thymic Tissues by “In Vivo Cryotechnique”
    Article Snippet: The immunostained sections were additionally incubated with biotin-conjugated rabbit polyclonal anti-goat IgG antibody or biotin-conjugated goat polyclonal anti-rabbit IgG antibody (Vector, Burlingame, CA, USA) at room temperature for 1 hr. .. The immunoreaction products were visualized with the avidin–biotin–HRP complex (ABC) method (Vector) and a DAB substrate kit (Pierce, Rockford, IL, USA), and additionally fixed with 0.05% osmium tetroxide solution, as reported previously [ , ]. .. All immunostained sections were counterstained with methyl green, and finally observed with a light microscope (BX-61; Olympus, Tokyo, Japan) ( p).

    Immunohistochemistry:

    Article Title: Synergistic suppression effect on tumor growth of acute myeloid leukemia by combining cytarabine with an engineered oncolytic vaccinia virus
    Article Snippet: After 25 days, the mice were sacrificed, and the tumors were excised, treated with 4% paraformaldehyde, and encased in paraffin for hematoxylin & eosin (H & E) staining, immunohistochemistry, and TUNEL assay. .. For the immunohistochemical (IHC) study, the sections were stained with anti-Caspase-3 or anti-ING4 (1:100 dilutions) primary antibodies overnight at 4°C, added with the avidin-biotin-peroxidase complex, and the slides were detected with diaminobenzidine Kit (Thermo Fisher Scientific). .. Images were photographed by an inverted fluorescence microscope (Nikon E300; Tokyo, Japan).

    Staining:

    Article Title: Synergistic suppression effect on tumor growth of acute myeloid leukemia by combining cytarabine with an engineered oncolytic vaccinia virus
    Article Snippet: After 25 days, the mice were sacrificed, and the tumors were excised, treated with 4% paraformaldehyde, and encased in paraffin for hematoxylin & eosin (H & E) staining, immunohistochemistry, and TUNEL assay. .. For the immunohistochemical (IHC) study, the sections were stained with anti-Caspase-3 or anti-ING4 (1:100 dilutions) primary antibodies overnight at 4°C, added with the avidin-biotin-peroxidase complex, and the slides were detected with diaminobenzidine Kit (Thermo Fisher Scientific). .. Images were photographed by an inverted fluorescence microscope (Nikon E300; Tokyo, Japan).

    Isolation:

    Article Title: Platelet-Derived Growth Factor Subunit B Signaling Promotes Pericyte Migration in Response to Loud Sound in the Cochlear Stria Vascularis
    Article Snippet: .. Cochleae were isolated and fixed with phosphate-buffered 3 % glutaraldehyde and 1.5 % PFA for 5 h. Stria vascularis from control and loud sound-stimulated groups were isolated and incubated in reaction buffer (Pierce™ DAB Substrate Kit, 34002, Thermo Fisher Scientific, USA) containing DAB and stable peroxide substrate for 1 h. After chemical reaction, samples were post-fixed for 1 h in 1 % osmium. .. Tissues were dehydrated through a graded alcohol series, embedded in Embed 812 (Electron Microscopy Sciences, Hatfield, PA., USA), sectioned, stained for 2 min with UranyLess (Electron Microscopy Sciences, USA), and examined on an FEI Tecnai-12 BioTWIN transmission electron microscope.

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    Thermo Fisher dab substrate kit
    Cellular organization of the adult thoracic NG. (A-C): Adult VNSs immunostained with anti-BRP (neuropil marker, blue) (A1-C4) and anti-Dll (NG marker, red) (B1-B3) with astrocytes expressing mCD8::GFP (membrane marker, green) (A1-C4), H2B::RFP (Nuclear marker, red) (B1-B3) or the multicolor system FB1.1 (single cell marker, red, yellow and green) (C1-C4) under the control of alrm-Gal4 . (B1-B3) , astrocyte (H2B::RFP+, Dll+): arrow, EG (Dll+): arrowhead. (C1-C4) Different astrocytes with processes occupying different neuropil territories: arrows. See also Figure S1 and video 1. (D-F): Adult VNSs immunostained with anti-BRP (neuropil marker, blue) (D1-F4) and anti-Dll (NG marker, red) ( E1-E3) with EG expressing mCD4::GFP (membrane marker, green) (D1-D3), H2B::RFP (nuclear marker, red) (E1-E3) or the multicolor system FB1.1 (single cell marker, red, yellow and green; ( Hadjieconomou et al., 2011 )) (F1-F4) under the control of R56F03-Gal4 . (E1-E3), astrocyte: arrow (Dll+), EG (H2B::RFP+, Dll+): arrowhead. (F1-F4) Different EG with processes occupying different neuropil territories: arrows. See also Figure S1 and video 1. (G-I): Prothoracic neuromeres with EG expressing mCD4::GFP and immunostained with anti-BRP (blue) and anti-Elav (neuron marker, red) (G) , anti-BRP (blue) and Dll (red) (H) or anti-NCad (neuropil marker, red) and Nrg (axon marker, blue) (I1-2) . (G) Asterisk: Elav+ neuron wrapped by an EG. (H) arrow: in more than half of the samples analyzed (N=10) a cell body of an EG (GFP +, Dll+) was observed inside the neuropil, next to axon bundles. Enlargements of the boxed regions are to the right of each panel. See also video 2. (J-K): Prothoracic neuromeres with EG expressing mCD4::GFP (J) and <t>GFP::mCD8::HRP</t> (K) immunostained with anti-BRP (blue) (J) or labeled with <t>DAB</t> (Black) (K) . (L) : Low magnification electron microscopy image of the boxed region in (K) . (M-P): enlargement of the boxed regions in (L) . PG: perineurial glia. SPG: subperineurial glia, NG: Neuropil Glia, C: Cortex, Np: Neuropil, NL: neural lamella. (O) : Left, schematic of adult CNS (blue: neuropils, grey: cortex); right, an individual astrocyte and EG labeled with mCD8::GFP under the control of alrm-Gal4 and R56F03-Gal4 using the MARCM technique. Axes: green (posterior), blue (dorsal), red (medial). (R): Average number of NG in the adult VNS, in which EG or astrocytes were expressing H2B::RFP under the control of R56F03-Gal4 or alrm-Gal4 , respectively, and immunostained with anti-Dll. Number of samples = 4/genotype. Error bars indicate standard deviation. ProNm: Prothoracic neuromere, AMesoNm: Accessory mesothoracic neuromere, MesoNm: Mesothoracic neuromere, MetaNm: Metathoracic neuromere, ANm: Abdominal neuromeres; FS: frontal cross section, TS: transverse cross section, 3D: 3 dimensional reconstruction of confocal image stack. pMP: partial maximum projection.
    Dab Substrate Kit, supplied by Thermo Fisher, 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/dab substrate kit/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dab substrate kit - by Bioz Stars, 2021-05
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    95
    Thermo Fisher metal enhanced diaminobenzidine
    Immunohistochemical staining for human Bcl-2 in thymi and spleens from Bcl-2 transgenic mice. Splenocytes that have a brown color (resulting from <t>diaminobenzidine</t> staining) are positive for human Bcl-2. Note the much greater staining in spleen vs. thymus in the Bcl-2 transgenic mice ( Top vs. Middle ). Although there was much less staining in thymi vs. spleens of Bcl-2 transgenics, the cells that were positive for Bcl-2 in thymi were resistant to sepsis-induced apoptosis. As is apparent in Bottom , none of the Bcl-2-positive cells in the septic thymus were apoptotic, i.e., none of the Bcl-2-positive cells had features of pyknosis or karyorrhexis ( n = 10 thymi from septic Bcl-2-Ig transgenic mice)
    Metal Enhanced Diaminobenzidine, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 1 article reviews
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    metal enhanced diaminobenzidine - by Bioz Stars, 2021-05
    95/100 stars
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    Image Search Results


    Cellular organization of the adult thoracic NG. (A-C): Adult VNSs immunostained with anti-BRP (neuropil marker, blue) (A1-C4) and anti-Dll (NG marker, red) (B1-B3) with astrocytes expressing mCD8::GFP (membrane marker, green) (A1-C4), H2B::RFP (Nuclear marker, red) (B1-B3) or the multicolor system FB1.1 (single cell marker, red, yellow and green) (C1-C4) under the control of alrm-Gal4 . (B1-B3) , astrocyte (H2B::RFP+, Dll+): arrow, EG (Dll+): arrowhead. (C1-C4) Different astrocytes with processes occupying different neuropil territories: arrows. See also Figure S1 and video 1. (D-F): Adult VNSs immunostained with anti-BRP (neuropil marker, blue) (D1-F4) and anti-Dll (NG marker, red) ( E1-E3) with EG expressing mCD4::GFP (membrane marker, green) (D1-D3), H2B::RFP (nuclear marker, red) (E1-E3) or the multicolor system FB1.1 (single cell marker, red, yellow and green; ( Hadjieconomou et al., 2011 )) (F1-F4) under the control of R56F03-Gal4 . (E1-E3), astrocyte: arrow (Dll+), EG (H2B::RFP+, Dll+): arrowhead. (F1-F4) Different EG with processes occupying different neuropil territories: arrows. See also Figure S1 and video 1. (G-I): Prothoracic neuromeres with EG expressing mCD4::GFP and immunostained with anti-BRP (blue) and anti-Elav (neuron marker, red) (G) , anti-BRP (blue) and Dll (red) (H) or anti-NCad (neuropil marker, red) and Nrg (axon marker, blue) (I1-2) . (G) Asterisk: Elav+ neuron wrapped by an EG. (H) arrow: in more than half of the samples analyzed (N=10) a cell body of an EG (GFP +, Dll+) was observed inside the neuropil, next to axon bundles. Enlargements of the boxed regions are to the right of each panel. See also video 2. (J-K): Prothoracic neuromeres with EG expressing mCD4::GFP (J) and GFP::mCD8::HRP (K) immunostained with anti-BRP (blue) (J) or labeled with DAB (Black) (K) . (L) : Low magnification electron microscopy image of the boxed region in (K) . (M-P): enlargement of the boxed regions in (L) . PG: perineurial glia. SPG: subperineurial glia, NG: Neuropil Glia, C: Cortex, Np: Neuropil, NL: neural lamella. (O) : Left, schematic of adult CNS (blue: neuropils, grey: cortex); right, an individual astrocyte and EG labeled with mCD8::GFP under the control of alrm-Gal4 and R56F03-Gal4 using the MARCM technique. Axes: green (posterior), blue (dorsal), red (medial). (R): Average number of NG in the adult VNS, in which EG or astrocytes were expressing H2B::RFP under the control of R56F03-Gal4 or alrm-Gal4 , respectively, and immunostained with anti-Dll. Number of samples = 4/genotype. Error bars indicate standard deviation. ProNm: Prothoracic neuromere, AMesoNm: Accessory mesothoracic neuromere, MesoNm: Mesothoracic neuromere, MetaNm: Metathoracic neuromere, ANm: Abdominal neuromeres; FS: frontal cross section, TS: transverse cross section, 3D: 3 dimensional reconstruction of confocal image stack. pMP: partial maximum projection.

    Journal: bioRxiv

    Article Title: Differing strategies used by motor neurons and glia to achieve robust development of an adult neuropil in Drosophila

    doi: 10.1101/149229

    Figure Lengend Snippet: Cellular organization of the adult thoracic NG. (A-C): Adult VNSs immunostained with anti-BRP (neuropil marker, blue) (A1-C4) and anti-Dll (NG marker, red) (B1-B3) with astrocytes expressing mCD8::GFP (membrane marker, green) (A1-C4), H2B::RFP (Nuclear marker, red) (B1-B3) or the multicolor system FB1.1 (single cell marker, red, yellow and green) (C1-C4) under the control of alrm-Gal4 . (B1-B3) , astrocyte (H2B::RFP+, Dll+): arrow, EG (Dll+): arrowhead. (C1-C4) Different astrocytes with processes occupying different neuropil territories: arrows. See also Figure S1 and video 1. (D-F): Adult VNSs immunostained with anti-BRP (neuropil marker, blue) (D1-F4) and anti-Dll (NG marker, red) ( E1-E3) with EG expressing mCD4::GFP (membrane marker, green) (D1-D3), H2B::RFP (nuclear marker, red) (E1-E3) or the multicolor system FB1.1 (single cell marker, red, yellow and green; ( Hadjieconomou et al., 2011 )) (F1-F4) under the control of R56F03-Gal4 . (E1-E3), astrocyte: arrow (Dll+), EG (H2B::RFP+, Dll+): arrowhead. (F1-F4) Different EG with processes occupying different neuropil territories: arrows. See also Figure S1 and video 1. (G-I): Prothoracic neuromeres with EG expressing mCD4::GFP and immunostained with anti-BRP (blue) and anti-Elav (neuron marker, red) (G) , anti-BRP (blue) and Dll (red) (H) or anti-NCad (neuropil marker, red) and Nrg (axon marker, blue) (I1-2) . (G) Asterisk: Elav+ neuron wrapped by an EG. (H) arrow: in more than half of the samples analyzed (N=10) a cell body of an EG (GFP +, Dll+) was observed inside the neuropil, next to axon bundles. Enlargements of the boxed regions are to the right of each panel. See also video 2. (J-K): Prothoracic neuromeres with EG expressing mCD4::GFP (J) and GFP::mCD8::HRP (K) immunostained with anti-BRP (blue) (J) or labeled with DAB (Black) (K) . (L) : Low magnification electron microscopy image of the boxed region in (K) . (M-P): enlargement of the boxed regions in (L) . PG: perineurial glia. SPG: subperineurial glia, NG: Neuropil Glia, C: Cortex, Np: Neuropil, NL: neural lamella. (O) : Left, schematic of adult CNS (blue: neuropils, grey: cortex); right, an individual astrocyte and EG labeled with mCD8::GFP under the control of alrm-Gal4 and R56F03-Gal4 using the MARCM technique. Axes: green (posterior), blue (dorsal), red (medial). (R): Average number of NG in the adult VNS, in which EG or astrocytes were expressing H2B::RFP under the control of R56F03-Gal4 or alrm-Gal4 , respectively, and immunostained with anti-Dll. Number of samples = 4/genotype. Error bars indicate standard deviation. ProNm: Prothoracic neuromere, AMesoNm: Accessory mesothoracic neuromere, MesoNm: Mesothoracic neuromere, MetaNm: Metathoracic neuromere, ANm: Abdominal neuromeres; FS: frontal cross section, TS: transverse cross section, 3D: 3 dimensional reconstruction of confocal image stack. pMP: partial maximum projection.

    Article Snippet: The expression of the HRP (Horseradish Peroxydase) were revealed overnight using the DAB substrate kit from Thermofisher.

    Techniques: Marker, Expressing, Labeling, Electron Microscopy, Standard Deviation

    Cellular Organization of the Adult Thoracic NG (A–C) Adult VNCs immunostained with anti-BRP (neuropil marker, blue) (A1–C) and anti-Dll (NG marker, green) (B) with astrocytes expressing mCD8::GFP (membrane marker, green) (A1 and A2), H2B::RFP (nuclear marker, red) (B) or the multicolor system FB1.1 (single-cell marker, red, yellow, and green) (C) under the control of alrm-Gal4 . (B) Astrocyte (H2B::RFP + , Dll + ): arrow, EG (Dll + ): arrowhead. . ) (F) under the control of R56F03-Gal4 . (E) Astrocyte: arrow (Dll + ), EG (H2B::RFP + , Dll + ): arrowhead. (D) arrow: in more than half of the samples analyzed (n = 10) a cell body of an EG (GFP + , Dll + ) was observed inside the neuropil, next to axon bundles. . (G and H) Prothoracic neuromeres with EG-expressing mCD4::GFP and immunostained with anti-BRP (blue) and anti-Elav (neuron marker, red) (G) or anti-NCad (neuropil marker, red) and Nrg (axon marker, blue) (H). (G) Asterisk: Elav + . (I and J) Prothoracic neuromeres with EG-expressing mCD4::GFP (I) and GFP::mCD8::HRP (J) immunostained with anti-BRP (blue) (I) or labeled with DAB (brown) (J). (K) Low-magnification electron microscope image of the boxed region in (J). (L–O) Enlargement of the boxed regions in (K). (L2) Enlargement of the boxed region in (L1). Note: (L1) and (L2) show the organization of the EG processes around the neuropil while (M)–(O) show the EG processes inside the neuropil. Pink lines outline axons. PG, perineurial glia; SPG, subperineurial glia; NG, neuropil glia; C, cortex; Np, neuropil; NL, neural lamella. (P) Bottom, schematic of adult CNS (blue: neuropils, gray: cortex); top, single astrocyte and EG labeled with mCD8::GFP under the control of alrm-Gal4 and R56F03-Gal4 using MARCM. Axes: green (posterior), blue (dorsal), red (medial). (Q) Average number of NG in the adult VNC, in which EG or astrocytes were expressing H2B::RFP under the control of R56F03-Gal4 or alrm-Gal4 , respectively, and immunostained with anti-Dll. Number of samples = 4/genotype. Error bars indicate SD. ProNm, Prothoracic neuromere; AMesoNm, Accessory mesothoracic neuromere; MesoNm, Mesothoracic neuromere; MetaNm, Metathoracic neuromere; ANm, Abdominal neuromeres; FS, frontal cross-section; TS, transverse cross-section; 3D, 3-dimensional reconstruction of confocal image stack; pMP, partial maximum projection.

    Journal: Neuron

    Article Title: Differing Strategies Despite Shared Lineages of Motor Neurons and Glia to Achieve Robust Development of an Adult Neuropil in Drosophila

    doi: 10.1016/j.neuron.2018.01.007

    Figure Lengend Snippet: Cellular Organization of the Adult Thoracic NG (A–C) Adult VNCs immunostained with anti-BRP (neuropil marker, blue) (A1–C) and anti-Dll (NG marker, green) (B) with astrocytes expressing mCD8::GFP (membrane marker, green) (A1 and A2), H2B::RFP (nuclear marker, red) (B) or the multicolor system FB1.1 (single-cell marker, red, yellow, and green) (C) under the control of alrm-Gal4 . (B) Astrocyte (H2B::RFP + , Dll + ): arrow, EG (Dll + ): arrowhead. . ) (F) under the control of R56F03-Gal4 . (E) Astrocyte: arrow (Dll + ), EG (H2B::RFP + , Dll + ): arrowhead. (D) arrow: in more than half of the samples analyzed (n = 10) a cell body of an EG (GFP + , Dll + ) was observed inside the neuropil, next to axon bundles. . (G and H) Prothoracic neuromeres with EG-expressing mCD4::GFP and immunostained with anti-BRP (blue) and anti-Elav (neuron marker, red) (G) or anti-NCad (neuropil marker, red) and Nrg (axon marker, blue) (H). (G) Asterisk: Elav + . (I and J) Prothoracic neuromeres with EG-expressing mCD4::GFP (I) and GFP::mCD8::HRP (J) immunostained with anti-BRP (blue) (I) or labeled with DAB (brown) (J). (K) Low-magnification electron microscope image of the boxed region in (J). (L–O) Enlargement of the boxed regions in (K). (L2) Enlargement of the boxed region in (L1). Note: (L1) and (L2) show the organization of the EG processes around the neuropil while (M)–(O) show the EG processes inside the neuropil. Pink lines outline axons. PG, perineurial glia; SPG, subperineurial glia; NG, neuropil glia; C, cortex; Np, neuropil; NL, neural lamella. (P) Bottom, schematic of adult CNS (blue: neuropils, gray: cortex); top, single astrocyte and EG labeled with mCD8::GFP under the control of alrm-Gal4 and R56F03-Gal4 using MARCM. Axes: green (posterior), blue (dorsal), red (medial). (Q) Average number of NG in the adult VNC, in which EG or astrocytes were expressing H2B::RFP under the control of R56F03-Gal4 or alrm-Gal4 , respectively, and immunostained with anti-Dll. Number of samples = 4/genotype. Error bars indicate SD. ProNm, Prothoracic neuromere; AMesoNm, Accessory mesothoracic neuromere; MesoNm, Mesothoracic neuromere; MetaNm, Metathoracic neuromere; ANm, Abdominal neuromeres; FS, frontal cross-section; TS, transverse cross-section; 3D, 3-dimensional reconstruction of confocal image stack; pMP, partial maximum projection.

    Article Snippet: HRP expression was revealed overnight using the DAB substrate kit (Thermofisher).

    Techniques: Marker, Expressing, Labeling, Microscopy

    Immunohistochemical staining for human Bcl-2 in thymi and spleens from Bcl-2 transgenic mice. Splenocytes that have a brown color (resulting from diaminobenzidine staining) are positive for human Bcl-2. Note the much greater staining in spleen vs. thymus in the Bcl-2 transgenic mice ( Top vs. Middle ). Although there was much less staining in thymi vs. spleens of Bcl-2 transgenics, the cells that were positive for Bcl-2 in thymi were resistant to sepsis-induced apoptosis. As is apparent in Bottom , none of the Bcl-2-positive cells in the septic thymus were apoptotic, i.e., none of the Bcl-2-positive cells had features of pyknosis or karyorrhexis ( n = 10 thymi from septic Bcl-2-Ig transgenic mice)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Prevention of lymphocyte cell death in sepsis improves survival in mice

    doi:

    Figure Lengend Snippet: Immunohistochemical staining for human Bcl-2 in thymi and spleens from Bcl-2 transgenic mice. Splenocytes that have a brown color (resulting from diaminobenzidine staining) are positive for human Bcl-2. Note the much greater staining in spleen vs. thymus in the Bcl-2 transgenic mice ( Top vs. Middle ). Although there was much less staining in thymi vs. spleens of Bcl-2 transgenics, the cells that were positive for Bcl-2 in thymi were resistant to sepsis-induced apoptosis. As is apparent in Bottom , none of the Bcl-2-positive cells in the septic thymus were apoptotic, i.e., none of the Bcl-2-positive cells had features of pyknosis or karyorrhexis ( n = 10 thymi from septic Bcl-2-Ig transgenic mice)

    Article Snippet: After rinsing, a secondary rat anti-mouse Ab (PharMingen, catalog no. M031737) (10 μl/ml) was added at room temperature for 30 min. After rinsing, a strepavidin-biotin complex (VECTASTAIN ABC; Vector Laboratories) was applied at room temperature for 30 min. After rinsing, metal-enhanced diaminobenzidine (Pierce) was applied.

    Techniques: Immunohistochemistry, Staining, Transgenic Assay, Mouse Assay

    Internalization rates of carboxyfluorescein-Ad5, -Ad7, and -Ad5f7. Carboxyfluorescein-Ad (10 11 particles/ml) was incubated with A549 cells at 4°C to evaluate only surface binding or at 37°C to allow binding and internalization of Ad. At each time point, cells were washed, fixed, and treated with HRP and DAB, with or without H 2 O 2 . Extracellular fluorophore was quenched by precipitation of DAB by HRP in the presence of H 2 O 2 . (A) A549 cells incubated with carboxyfluorescein-Ad5 (4°C for 1 h) and treated with HRP-DAB without H 2 O 2 . (B) Same as panel A, but treated with HRP-DAB in the presence of H 2 O 2 . (C) A549 cells infected with carboxyfluorescein-Ad5 at 37°C for 10 min, washed, and incubated for an additional 10 min at 37°C, then treated with HRP-DAB without H 2 O 2 . (D) Same as panel C, but treated with HRP-DAB in the presence of H 2 O 2 . (E) A549 cells incubated with carboxyfluorescein-Ad7 (4°C for 1 h) and treated with HRP-DAB without H 2 O 2 . (F) Same as panel E, but treated with HRP-DAB in the presence of H 2 O 2 . (G) A549 cells infected with carboxyfluorescein-Ad7 at 37°C for 10 min, washed, and incubated for an additional 10 min at 37°C, then treated with HRP-DAB without H 2 O 2 . (H) Same as panel G, but treated with HRP-DAB in the presence of H 2 O 2 . (I) A549 cells incubated with carboxyfluorescein-Ad5f7 (4°C for 1 h) and treated with HRP-DAB without H 2 O 2 . (J) Same as panel I, but treated with HRP-DAB in the presence of H 2 O 2 . (K) A549 cells infected with carboxyfluorescein-Ad5f7 at 37°C for 10 min, washed, and incubated for an additional 10 min at 37°C, then treated with HRP-DAB without H 2 O 2 . (L) Same as panel K, but treated with HRP-DAB in the presence of H 2 O 2 . Bar = 10 μm. (M) Percentage of internalized Ad per cell relative to the total amount of Ad per cell. Levels of internalized Ad5 (●), Ad7 (○), and Ad5f7 (▵) were determined by digital image analysis (see Materials and Methods). Data for zero internalization time correspond to surface-labeled cells (4°C binding for 1 h).

    Journal: Journal of Virology

    Article Title: Fiber Swap between Adenovirus Subgroups B and C Alters Intracellular Trafficking of Adenovirus Gene Transfer Vectors

    doi:

    Figure Lengend Snippet: Internalization rates of carboxyfluorescein-Ad5, -Ad7, and -Ad5f7. Carboxyfluorescein-Ad (10 11 particles/ml) was incubated with A549 cells at 4°C to evaluate only surface binding or at 37°C to allow binding and internalization of Ad. At each time point, cells were washed, fixed, and treated with HRP and DAB, with or without H 2 O 2 . Extracellular fluorophore was quenched by precipitation of DAB by HRP in the presence of H 2 O 2 . (A) A549 cells incubated with carboxyfluorescein-Ad5 (4°C for 1 h) and treated with HRP-DAB without H 2 O 2 . (B) Same as panel A, but treated with HRP-DAB in the presence of H 2 O 2 . (C) A549 cells infected with carboxyfluorescein-Ad5 at 37°C for 10 min, washed, and incubated for an additional 10 min at 37°C, then treated with HRP-DAB without H 2 O 2 . (D) Same as panel C, but treated with HRP-DAB in the presence of H 2 O 2 . (E) A549 cells incubated with carboxyfluorescein-Ad7 (4°C for 1 h) and treated with HRP-DAB without H 2 O 2 . (F) Same as panel E, but treated with HRP-DAB in the presence of H 2 O 2 . (G) A549 cells infected with carboxyfluorescein-Ad7 at 37°C for 10 min, washed, and incubated for an additional 10 min at 37°C, then treated with HRP-DAB without H 2 O 2 . (H) Same as panel G, but treated with HRP-DAB in the presence of H 2 O 2 . (I) A549 cells incubated with carboxyfluorescein-Ad5f7 (4°C for 1 h) and treated with HRP-DAB without H 2 O 2 . (J) Same as panel I, but treated with HRP-DAB in the presence of H 2 O 2 . (K) A549 cells infected with carboxyfluorescein-Ad5f7 at 37°C for 10 min, washed, and incubated for an additional 10 min at 37°C, then treated with HRP-DAB without H 2 O 2 . (L) Same as panel K, but treated with HRP-DAB in the presence of H 2 O 2 . Bar = 10 μm. (M) Percentage of internalized Ad per cell relative to the total amount of Ad per cell. Levels of internalized Ad5 (●), Ad7 (○), and Ad5f7 (▵) were determined by digital image analysis (see Materials and Methods). Data for zero internalization time correspond to surface-labeled cells (4°C binding for 1 h).

    Article Snippet: After infection at 4°C or infection with incubation at 37°C as indicated, the cells were washed three times with ice-cold PBS and incubated with HRP (10 μg/ml; Pierce, Rockford, Ill.) and metal-enhanced DAB (Pierce) in PBS or in stable peroxide buffer (H2 O2 ; Pierce) for 1 h at 4°C.

    Techniques: Incubation, Binding Assay, Infection, Labeling