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  • 99
    Thermo Fisher nbt bcip
    Double immunocytochemistry in CA1 of the hippocampus with CP-3 ( A , C , and E ; <t>NBT/BCIP</t> in blue ). A, AKAP79; C, PKA-Cβ; E, PKA-RII (DAB in brown ). PG-5 staining ( B , D , and F ; NBT/BCIP in blue ); B, AKAP79; D, PKA-Cβ; F, PKA-RIIβ (DAB in brown ). Note the colocalization of CP-3 and PG-5 with AKAP79-, PKA-RIIβ-, and PKA-Cβ-reactive neurons.
    Nbt Bcip, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 721 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore stock solution
    Double immunocytochemistry in CA1 of the hippocampus with CP-3 ( A , C , and E ; <t>NBT/BCIP</t> in blue ). A, AKAP79; C, PKA-Cβ; E, PKA-RII (DAB in brown ). PG-5 staining ( B , D , and F ; NBT/BCIP in blue ); B, AKAP79; D, PKA-Cβ; F, PKA-RIIβ (DAB in brown ). Note the colocalization of CP-3 and PG-5 with AKAP79-, PKA-RIIβ-, and PKA-Cβ-reactive neurons.
    Stock Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 902 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bcip nbt
    Inhibition of LCMV gene expression in IFN-treated PML +/+ and PML −/− MEF. (A) Cells were treated for 48 h with 500, 1,000, or 1,500 U of murine IFN-γ per ml, trypsinized, replated into equivalent pools, and then infected with LCMV at an MOI of 1 PFU for 24 h. Western blot analysis of the MEF extracts was done as described in the text. Blots were probed with our guinea pig anti-LCMV antibodies and revealed by <t>BCIP-NBT,</t> an alkaline phosphatase substrate. (B) The experiment in panel A was repeated; however, Western blots were developed with a chemiluminescent probe and scanned with a PhosphorImager (see the text). Scanning allowed us to attribute values (the AQR) to the band intensities of the viral NP and to normalize these values to an internal standard, murine actin. We considered the amount of NP expressed by untreated PML −/− cells to be 100%, and we presented the NP of all other cells as a percentage of this value.
    Bcip Nbt, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1519 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Beyotime bcip nbt alkaline phosphatase color development kit
    The effects of hPTH(1–34) and GR(1–34) on osteogenesis and osteoclastogenesis in bone marrow cells of ORX mice. At the 2nd week of administration, ALP activity of the cells in osteogenic medium were stained and measured with a <t>BCIP/NBT</t> Color Development Kit ( a ); the mineralized nodules were stained with Alizarin Red S solution and calcium deposition was quantified spectrophotometrically ( b ). At the 1st week of administration, TRAP staining was performed and the percentage of TRAP+ cells to total cells were counted ( c ). [Three independent experiments were repeated for ( a b ) and six for ( c ); 10 fields at 100 magnitude were randomly selected for TRAP+ cell counting; * P
    Bcip Nbt Alkaline Phosphatase Color Development Kit, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 980 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bcip
    SDS-PAGE and Western blot of rJpIocys2a, rBrBmcys2b and rBrBmcys2c production. Western blot: purified rGST-JpIocys2a probed with anti-GST-Hl primary antibody and rabbit anti-IgG secondary antibody conjugate with alkaline phosphatase; purified rBrBmcys2b and rBrBmcys2c probed with anti-histidine tag primary antibody conjugate with alkaline phosphatase. Alkaline phosphatase revelations were performed with <t>NBT</t> and <t>BCIP.</t> SDS-PAGE: Recombinant cystatins resolved by 14% SDS-PAGE were stained with Coomassie blue G-250; purified rGST-JpIocys2a before and after thrombin cleavage (rGST and JpIocys2a); purified rJpIocys2a, rBrBmcys2b and rBrBmcys2c. MW: molecular weight.
    Bcip, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1095 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore nbt bcip
    Histone H2AX phosphorylated at S139 (H2AXS139Ph) is a marker of double-stranded breaks (DSBs), while poly(ADP-ribose) polymerase-2 (PARP-2) is a marker of single-stranded breaks (SSBs). (A) Results of the immunocytochemical analysis and the method of tissue printing . (a-a', b-c) the presentation of superimposed fluorescence images (DAPI-related in blue and H2AXS139Ph-related in green) after the immunocytochemical detection of H2AXS139Ph in (a) control, (b) after 2.5 mM hydroxyurea-treatment (HU) for 32 h, (c) after 24-h synchronization under the influence of 2.5 mM HU and 8-h co-treatment with 2.5 mM HU and 5 mM caffeine (CF). (a') negative control; incubation exclusively with secondary antibodies. The values of marking indices (expressed in percents) are presented in the top left corner on the following images: (a)–for control series; (b)–after 32-h treatment with HU; (c)–after the induction of premature chromosome condensation (PCC) under the influence of HU/CF. Scale bars in a-a', b-c are 20 μm. (d-f) identification of H2AXS139Ph in the top sections of Vicia faba roots by the method of tissue printing , negative images. In the top left corner of each negative image, there is a miniature of the same fragment of nitrocellulose membrane in color, i.e. stained in the reaction of <t>NBT/BCIP</t> (d'-f'). (d-d') control, (e-e') HU, 32 h, (f-f') HU for 24 h and co-incubation HU/CF for 8 h (total incubation time: 32 h). Scale bars in d'-f' and d-f are 10 mm. (g-g', h-i) presentation of superimposed fluorescence images (DAPI-related in blue and PARP-2-related in green) after the immunocytochemical detection of PARP-2: (g) control, (h) after HU-treatment for 32 h, (i) after 24-h synchronization under the influence HU and 8-h co-treatment with HU/CF. (g') negative control; incubation exclusively with secondary antibodies. The values of marking (expressed in percents) are presented in the top left corner of the following images (g) control series; (h) after 32-h treatment with HU; (i) after the induction of PCC under the influence of HU/CF. Scale bars in g-g', h-i are 20 μm. (j-l) identification of PARP-2 in the top section of V . faba roots by the method of tissue printing , negative images. In the top left corner of each negative image, there is a miniature of the same fragment of nitrocellulose membrane in color, i.e. stained in the reaction of NBT/BCIP (j'-l'). (j-j') control, (k-k') HU, 32 h, (l-l') HU for 24 h and co-incubation HU/CF. Scale bars in j'-l' and j-l are 10 mm. (B) Identification of proteins H2AXS139Ph and PARP-2 by the method of Western blot. (a-a') expression levels of the H2AXS139Ph by Western blot analysis. Data shown are the representatives of three independent experiments. The relative levels of H2AXS139Ph after normalization for actin, as determined by densitometry analysis of the bands, are shown in the histogram (a'; the pixel values [ pv ; 1–255] categorized according to densitometry analysis of the band intensities and expressed in arbitrary units [a.u.]). Columns , mean from three independent experiments; bars , SD. * p ≤ 0.001 (Control/HU, Mann-Whitney U test); ▲ p ≤ 0.01 (Control/PCC, Mann-Whitney U test). (b-b') expression levels of the PARP-2 by Western blot analysis. Data shown are representative of three independent experiments. The relative levels of PARP-2 after normalization for actin, as determined by densitometry analysis of the bands, are shown in the histogram (b'; the pixel values [ pv ; 1–255] categorized according to densitometry analysis of the band intensities and expressed in arbitrary units [a.u.]). Columns , mean from three independent experiments; bars , SD. ▲ p ≤ 0.01 (Control/HU, Mann-Whitney U test); * p ≤ 0.001 (Control/PCC, Mann-Whitney U test).
    Nbt Bcip, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bcip nbt substrate
    Immunoprecipitation and Western-blot analysis of DEL, KMH2 and L-428 cell lysates. 23.1 (Panel A and C) and R24.1 (Panel B and D). Samples were separated on 4–20% Life-gel and transferred onto nitrocellulose membranes. The membranes were then blocked in 5% non-fat dry milk and probed with monoclonal antibody clones R23.1 (Panel A and B) and R24.1 (Panel C and D) followed by goat-anti-mouse IgG-AP. The membranes were developed in <t>BCIP/NBT.</t> A protein band of ~21 KDa as well as IgG light chain were detected in all three lysates examined. Lane 1 contains molecular weights and lanes 2–4 contain Immunoprecipitated samples of DEL, KMH2 and L-428.
    Bcip Nbt Substrate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 611 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Promega bcip nbt color development substrate
    Titers and cross-reactivity of antivenoms raised against B . arietans and Bitis g . rhinoceros plus B . nasicornis venoms. [A] ELISA : ELISA plates were coated with 1.0 μg of the Bitis spp venoms/well and incubated with different dilutions of the horse experimental antivenoms produced against B . arietans venom (α-Ba antivenom), B . g . rhinoceros plus B . nasicornis venoms (α-Br + Bn antivenom) or with the botulinic toxin antiserum (control), followed by anti-horse IgG-HRPO-conjugated. The results were expressed as the mean of absorbance value ± SD. [B] Western Blot : venom samples (5 μg) from B . arietans (Ba), B . g . rhinoceros (Br) and B . nasicornis (Bn) snakes were separated by SDS-PAGE, electrotransfered to nitrocellulose membranes and incubated with the antivenoms raised against B . arietans (α-Ba), B . g . rhinoceros plus B . nasicornis (α-Br+Bn) or with botulinic toxin antiserum (control) diluted 1:2000 followed by GAH/Ig-AP. The reactions were revealed with <t>NBT</t> and <t>BCIP.</t>
    Bcip Nbt Color Development Substrate, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 506 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sigmafast bcip nbt
    Continuous exposure to gemcitabine increases tumorigenicity but continuous exposure to sulforaphane or quercetin reduces it. ( a ) BxPC-3, Bx-GEM, Bx-Q and Bx-SF cells were seeded at a low density (2000 cells/well) in 6-well plates. After 2 weeks, cells were Coomassie-stained and colonies containing more than 50 cells were counted under a dissecting microscope. The survival fraction and representative photographs of colonies (first generation) are presented on the left. For second-generation colony formation, an equal amount of living cells from first-generation colonies were collected and 2000 cells per well were re-seeded. The colony formation was analyzed as described above and is presented on the right. ( b ) Cells were cultured to 90% confluence before the cell layer was scratched with the tip of a 10- μ l pipette. Closure of the wounded region was evaluated 24 h after scratching by microscopy at × 100 magnification. For quantification of the scratched area, the percentage of the gap area was evaluated and calculated by TScratch software (diagram below photographs). ( c ) Cells were seeded in 6-well plates, followed by exposure to NH Osteo-Diff medium to induce osteocytic differentiation. Fourteen days later, the cells were stained with <t>BCIP/NBT</t> substrate for alkaline phosphatases, expressed by cells differentiated into osteocytes, which appear dark. Representative images at × 200 magnification are shown. ( d ) Proteins were harvested and the expression of EpCAM, Nanog, Twist2 and E-cadherin was measured by western blot analysis. β -Actin was used as a loading control. Three independent experiments were performed at least in triplicates and the data are presented as means ±S.D. * P
    Sigmafast Bcip Nbt, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 407 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher 1 step nbt bcip
    Dot blots showing binding of biotinylated fibrinogen by S. iniae . Strains QMA0076 and QMA0141 were incubated with biotinylated fibrinogen, washed and transferred to PVDF, then membranes were probed with streptavidin-alkaline phosphatase conjugate and developed with <t>NBT/BCIP.</t> Panel A) Lanes 1 and 2 comprised 5 μl cells grown in vegetable peptone, whilst lanes 2 and 3 contained 5 μl cells grown in barramundi serum. Cells were harvested and washed in TBS before being incubated with biotinylated human fibrinogen (Lanes 1 and 3) or TBS (lanes 2 and 4) for 20 min, prior to extensive washing and transfer onto PVDF membrane for subsequent detection. Fb indicates serial two-fold dilutions of biotinylated fibrinogen commencing at 2.5 μg/mL. Panel B) shows serial 2 × dilutions of 5 μl cells diluted in TBS. Lane F, cells incubated first with TBS then with biotinylated fibrinogen before transfer to the membrane. Lane P+F, cells incubated with barramundi plasma followed by biotinylated fibrinogen, Lane TBS, cells incubated twice in TBS.
    1 Step Nbt Bcip, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore bcip nbt liquid substrate system
    Establishment of transgenic cell lines stably expressing EGFP and mCherry (RFP). (a) A schematic illustration of the constructed ΔDE-pOG2-ΔPE-pOm2 vector using the porcine Oct4 promoter. DE: distal enhancer; PE: proximal enhancer. (b) Genotyping the Oct4 promoter-driven transgenes from genomic DNA. PC: positive control; DW: distilled water, negative control; WT: wild type; TG: transgenic cell lines. Lanes 3–6, fluorescence-positive cell lines established from wild type F9 cells, P19 cells, mESCs, and MEFs. (c) Reverse transcriptional PCR analysis of pluripotency markers in fluorescence-positive cell lines. (d) Surface alkaline phosphatase activity in transgenic F9 cells, P19 cells, and mESCs was measured using <t>BCIP/NBT.</t> (e) Typical pluripotency markers, Nanog and Oct4, were assayed by immunofluorescent cytochemical staining in transgenic F9 cells, P19 cells, and mESCs. Scale bars indicate 20 μ m.
    Bcip Nbt Liquid Substrate System, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 330 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Beyotime bcip nbt alp color development kit
    MiR-17-5p and miR-27b-3p were differentially expressed during osteogenic differentiation of human ligament fibroblasts ( A ) <t>ALP</t> in fibroblasts was stained using the <t>BCIP/NBT</t> kit after the cells were incubated in OM for 7 and 14 days. Scale bar = 200 μm. ( B ) The ALP activity of fibroblasts was measured after they were incubated in OM for 7 and 14 days. ( C ) Fibroblasts were incubated in OM for 21 days, and then the mineralized nodules were stained using alizarin red S (ARS). Scale bar = 200 μm. ( D ) Mineralization was quantified by extraction of ARS dye with 10% cetylpyridinium chloride. ( E ) The total RNA was isolated on days 7 and 14. Runx2, ALP, and COL1A1 mRNA levels were determined using qRT-PCR and normalized to GAPDH. ( F ) The total RNA was isolated on days 7 and 14. Levels of miR-17-5p and miR-27b-3p were determined using qRT-PCR and normalized to U6. * P
    Bcip Nbt Alp Color Development Kit, supplied by Beyotime, used in various techniques. Bioz Stars score: 89/100, based on 189 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher bcip
    MiR-17-5p and miR-27b-3p were differentially expressed during osteogenic differentiation of human ligament fibroblasts ( A ) <t>ALP</t> in fibroblasts was stained using the <t>BCIP/NBT</t> kit after the cells were incubated in OM for 7 and 14 days. Scale bar = 200 μm. ( B ) The ALP activity of fibroblasts was measured after they were incubated in OM for 7 and 14 days. ( C ) Fibroblasts were incubated in OM for 21 days, and then the mineralized nodules were stained using alizarin red S (ARS). Scale bar = 200 μm. ( D ) Mineralization was quantified by extraction of ARS dye with 10% cetylpyridinium chloride. ( E ) The total RNA was isolated on days 7 and 14. Runx2, ALP, and COL1A1 mRNA levels were determined using qRT-PCR and normalized to GAPDH. ( F ) The total RNA was isolated on days 7 and 14. Levels of miR-17-5p and miR-27b-3p were determined using qRT-PCR and normalized to U6. * P
    Bcip, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 300 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Double immunocytochemistry in CA1 of the hippocampus with CP-3 ( A , C , and E ; NBT/BCIP in blue ). A, AKAP79; C, PKA-Cβ; E, PKA-RII (DAB in brown ). PG-5 staining ( B , D , and F ; NBT/BCIP in blue ); B, AKAP79; D, PKA-Cβ; F, PKA-RIIβ (DAB in brown ). Note the colocalization of CP-3 and PG-5 with AKAP79-, PKA-RIIβ-, and PKA-Cβ-reactive neurons.

    Journal: The Journal of Neuroscience

    Article Title: cAMP-Dependent Protein Kinase Phosphorylations on Tau in Alzheimer’s Disease

    doi: 10.1523/JNEUROSCI.19-17-07486.1999

    Figure Lengend Snippet: Double immunocytochemistry in CA1 of the hippocampus with CP-3 ( A , C , and E ; NBT/BCIP in blue ). A, AKAP79; C, PKA-Cβ; E, PKA-RII (DAB in brown ). PG-5 staining ( B , D , and F ; NBT/BCIP in blue ); B, AKAP79; D, PKA-Cβ; F, PKA-RIIβ (DAB in brown ). Note the colocalization of CP-3 and PG-5 with AKAP79-, PKA-RIIβ-, and PKA-Cβ-reactive neurons.

    Article Snippet: Double-labeled sections were also incubated with alkaline phosphatase-labeled secondaries and reacted with NBT/BCIP (Pierce).

    Techniques: Immunocytochemistry, Staining

    Anti-NP B cells in ectopic lymphoid tissue A, Light chain staining of B cells in lipogranulomas from B6 mice treated with TMPD alone or treated with TMPD and then immunized with NP-KLH. Paraformaldehyde-fixed tissue was analyzed 12 days after NP-KLH immunization. Paraffin sections were stained with anti-κ and anti-λ light chain antibodies. B, Number of κ and λ positive cells per high power field in mice treated with TMPD + NP-KLH immunization or with TMPD alone (* P = 0.02, Mann Whitney test; representative of five independent experiments). C, ELISPOT assay for anti-NP B cells. MultiScreen HTS IP plates containing a 0.45 µm Immobilon-P membrane were coated with 1 µg/mL NP-BSA. Lipogranuloma and spleen cells from TMPD treated mice (n = 2) or TMPD-treated and NP-KLH immunized mice (n = 2) were added to triplicate wells for 24 h before adding biotinylated goat anti-mouse IgM antibodies, streptavidin-peroxidase, and BCIP-NBT substrate. Number of spots per well was determined (* P = 0.03 for both mouse A and mouse B, Mann Whitney test; representative of three independent experiments). D, Relative sizes of individual spots in ELISPOT assays using lipogranuloma (Lipo) or spleen (Spl) cells from TMPD treated mice either with or without NP-KLH immunization.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Co-localization of Antigen-specific B and T Cells within Ectopic Lymphoid Tissue following Immunization with Exogenous Antigen

    doi:

    Figure Lengend Snippet: Anti-NP B cells in ectopic lymphoid tissue A, Light chain staining of B cells in lipogranulomas from B6 mice treated with TMPD alone or treated with TMPD and then immunized with NP-KLH. Paraformaldehyde-fixed tissue was analyzed 12 days after NP-KLH immunization. Paraffin sections were stained with anti-κ and anti-λ light chain antibodies. B, Number of κ and λ positive cells per high power field in mice treated with TMPD + NP-KLH immunization or with TMPD alone (* P = 0.02, Mann Whitney test; representative of five independent experiments). C, ELISPOT assay for anti-NP B cells. MultiScreen HTS IP plates containing a 0.45 µm Immobilon-P membrane were coated with 1 µg/mL NP-BSA. Lipogranuloma and spleen cells from TMPD treated mice (n = 2) or TMPD-treated and NP-KLH immunized mice (n = 2) were added to triplicate wells for 24 h before adding biotinylated goat anti-mouse IgM antibodies, streptavidin-peroxidase, and BCIP-NBT substrate. Number of spots per well was determined (* P = 0.03 for both mouse A and mouse B, Mann Whitney test; representative of three independent experiments). D, Relative sizes of individual spots in ELISPOT assays using lipogranuloma (Lipo) or spleen (Spl) cells from TMPD treated mice either with or without NP-KLH immunization.

    Article Snippet: Spots were developed with BCIP/NBT (Pierce) and incubated overnight before counting using a dissecting microscope.

    Techniques: Staining, Mouse Assay, MANN-WHITNEY, Enzyme-linked Immunospot

    Inhibition of LCMV gene expression in IFN-treated PML +/+ and PML −/− MEF. (A) Cells were treated for 48 h with 500, 1,000, or 1,500 U of murine IFN-γ per ml, trypsinized, replated into equivalent pools, and then infected with LCMV at an MOI of 1 PFU for 24 h. Western blot analysis of the MEF extracts was done as described in the text. Blots were probed with our guinea pig anti-LCMV antibodies and revealed by BCIP-NBT, an alkaline phosphatase substrate. (B) The experiment in panel A was repeated; however, Western blots were developed with a chemiluminescent probe and scanned with a PhosphorImager (see the text). Scanning allowed us to attribute values (the AQR) to the band intensities of the viral NP and to normalize these values to an internal standard, murine actin. We considered the amount of NP expressed by untreated PML −/− cells to be 100%, and we presented the NP of all other cells as a percentage of this value.

    Journal: Journal of Virology

    Article Title: Role of the Promyelocytic Leukemia Protein PML in the Interferon Sensitivity of Lymphocytic Choriomeningitis Virus

    doi: 10.1128/JVI.75.13.6204-6208.2001

    Figure Lengend Snippet: Inhibition of LCMV gene expression in IFN-treated PML +/+ and PML −/− MEF. (A) Cells were treated for 48 h with 500, 1,000, or 1,500 U of murine IFN-γ per ml, trypsinized, replated into equivalent pools, and then infected with LCMV at an MOI of 1 PFU for 24 h. Western blot analysis of the MEF extracts was done as described in the text. Blots were probed with our guinea pig anti-LCMV antibodies and revealed by BCIP-NBT, an alkaline phosphatase substrate. (B) The experiment in panel A was repeated; however, Western blots were developed with a chemiluminescent probe and scanned with a PhosphorImager (see the text). Scanning allowed us to attribute values (the AQR) to the band intensities of the viral NP and to normalize these values to an internal standard, murine actin. We considered the amount of NP expressed by untreated PML −/− cells to be 100%, and we presented the NP of all other cells as a percentage of this value.

    Article Snippet: Blots were developed using BCIP-NBT (Sigma) or enhanced chemiluminescence (Pierce, Rockford, Ill.).

    Techniques: Inhibition, Expressing, Infection, Western Blot

    Activation of AMPK enhances FTO expression in C3H10T1/2 cells (A and B) C3H10T1/2 cells were transfected with pcDNA3.0-AMPK (2 μg) for 6 h and then incubated for a further 12 h. (A) RT-PCR analysis was performed using total RNA extracted from cells. (B) Western blot analysis was performed using the indicated antibodies. (C and D) C3H10T1/2 cells were treated with BMP2 (0.25 μg/ml) for 24 h. Com.C (+, 0.1 μM; ++, 1 μM) was added 3 h prior to harvest. (C) RT-PCR analysis was performed using total RNA extracted from cells. (D) Western blot analysis was performed using the indicated antibodies. (E) C3H10T1/2 cells were transiently transfected with pc-DNA3.0AMPK (0.4 μg/well) and/or treated with BMP2 (0.25 μg/ml) for 4 days, and then stained with BCIP/NBT liquid substrate. ** P

    Journal: Molecules and Cells

    Article Title: Fat Mass and Obesity-Associated (FTO) Stimulates Osteogenic Differentiation of C3H10T1/2 Cells by Inducing Mild Endoplasmic Reticulum Stress via a Positive Feedback Loop with p-AMPK

    doi: 10.14348/molcells.2019.0136

    Figure Lengend Snippet: Activation of AMPK enhances FTO expression in C3H10T1/2 cells (A and B) C3H10T1/2 cells were transfected with pcDNA3.0-AMPK (2 μg) for 6 h and then incubated for a further 12 h. (A) RT-PCR analysis was performed using total RNA extracted from cells. (B) Western blot analysis was performed using the indicated antibodies. (C and D) C3H10T1/2 cells were treated with BMP2 (0.25 μg/ml) for 24 h. Com.C (+, 0.1 μM; ++, 1 μM) was added 3 h prior to harvest. (C) RT-PCR analysis was performed using total RNA extracted from cells. (D) Western blot analysis was performed using the indicated antibodies. (E) C3H10T1/2 cells were transiently transfected with pc-DNA3.0AMPK (0.4 μg/well) and/or treated with BMP2 (0.25 μg/ml) for 4 days, and then stained with BCIP/NBT liquid substrate. ** P

    Article Snippet: Briefly, cultured cells were fixed with 10% formaldehyde, rinsed twice with deionized water, and treated with BCIP/NBT solution (Sigma Aldrich, USA) for 15 min.

    Techniques: Activation Assay, Expressing, Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining

    Characterization of human adipose-derived stem cells. (a) Human ASCs were characterized by flow cytometry based on the expression of mesenchymal stem cell (CD44, CD73, CD90, and CD105) and hematopoietic stem cell (CD14, CD34, and CD45) markers. The red histograms refer to the isotype controls, and the blue histograms refer to the human ASC samples for the test. (b) ASCs were differentiated into adipocytes, osteoblasts, and chondrocytes upon induction. Adipogenesis differentiation was evaluated by Oil Red O staining (red) and hematoxylin counterstain (blue), and the yellowish lipid depositions were observed in the differentiated cells. For the osteogenesis differentiation, the NBT/BCIP and Alizarin Red S staining demonstrated that the differentiated cells possess strong alkaline phosphatase activity (blue) and reddish calcium deposition, respectively. Chondrogenesis differentiation was evaluated by Alcian blue staining, and the production of acidic polysaccharide (light blue) can be observed in the differentiated cells. Scale bar: 100 μ m.

    Journal: Stem Cells International

    Article Title: Notch Signaling Activation Enhances Human Adipose-Derived Stem Cell Retinal Differentiation

    doi: 10.1155/2018/9201374

    Figure Lengend Snippet: Characterization of human adipose-derived stem cells. (a) Human ASCs were characterized by flow cytometry based on the expression of mesenchymal stem cell (CD44, CD73, CD90, and CD105) and hematopoietic stem cell (CD14, CD34, and CD45) markers. The red histograms refer to the isotype controls, and the blue histograms refer to the human ASC samples for the test. (b) ASCs were differentiated into adipocytes, osteoblasts, and chondrocytes upon induction. Adipogenesis differentiation was evaluated by Oil Red O staining (red) and hematoxylin counterstain (blue), and the yellowish lipid depositions were observed in the differentiated cells. For the osteogenesis differentiation, the NBT/BCIP and Alizarin Red S staining demonstrated that the differentiated cells possess strong alkaline phosphatase activity (blue) and reddish calcium deposition, respectively. Chondrogenesis differentiation was evaluated by Alcian blue staining, and the production of acidic polysaccharide (light blue) can be observed in the differentiated cells. Scale bar: 100 μ m.

    Article Snippet: For alkaline phosphatase activity detection, the cells were fixed in ice-cold acetone and incubated with NBT/BCIP solution for 20 min. For calcium deposition analysis, the cells were fixed in 4% paraformaldehyde and stained with Alizarin Red S (Sigma-Aldrich) for 2-3 min. Chondrogenesis differentiation of human ASCs was induced by the osteocyte/chondrocyte differentiation basal medium with 10% chondrocyte supplement (StemPro, Gibco) on a Matrigel coating for 14 days.

    Techniques: Derivative Assay, Flow Cytometry, Cytometry, Expressing, Staining, Activity Assay

    In vitro analysis of Cry1Aa toxin binding. 30 µg each of fat body, Malpighian tubule and salivary gland membrane protein fractions prepared from early fifth instar (5E) larvae were separated by 7.5% SDS-PAGE, transferred onto nitrocellulose membranes, then incubated with biotinylated Cry1Aa toxin (200 ng/ml), followed by incubation with streptavidin-ALP conjugate and finally developed with NBT-BCIP substrate. Note the detection of a 113 kDa interacting membrane protein in all the three tissues. Blots labeled ( − ) are the control blots incubated with unlabeled Cry1Aa toxin while those labeled (+) are the blots incubated with biotinylated Cry1Aa toxin.

    Journal: PLoS ONE

    Article Title: Functional Interpretation of a Non-Gut Hemocoelic Tissue Aminopeptidase N (APN) in a Lepidopteran Insect Pest Achaea janata

    doi: 10.1371/journal.pone.0079468

    Figure Lengend Snippet: In vitro analysis of Cry1Aa toxin binding. 30 µg each of fat body, Malpighian tubule and salivary gland membrane protein fractions prepared from early fifth instar (5E) larvae were separated by 7.5% SDS-PAGE, transferred onto nitrocellulose membranes, then incubated with biotinylated Cry1Aa toxin (200 ng/ml), followed by incubation with streptavidin-ALP conjugate and finally developed with NBT-BCIP substrate. Note the detection of a 113 kDa interacting membrane protein in all the three tissues. Blots labeled ( − ) are the control blots incubated with unlabeled Cry1Aa toxin while those labeled (+) are the blots incubated with biotinylated Cry1Aa toxin.

    Article Snippet: The pull-down Cry1Aa toxin-interacting proteins were separated by 7.5% SDS-PAGE, electro-blotted onto nitrocellulose membrane (Pall Life Sciences), incubated with A. janata fat body APN polyclonal antibody (1∶10000 dilutions) , followed by incubation with ALP-conjugated goat anti-rabbit IgG and finally developed with NBT-BCIP substrate (Sigma-Aldrich).

    Techniques: In Vitro, Binding Assay, SDS Page, Incubation, ALP Assay, Labeling

    In vitro Cry1Aa toxin interaction in AjAPN1 knockdown larvae. Fat body (Fb), Malpighian tubule (Mt) and salivary gland (Sg) membrane protein fractions (30 µg each) of target and control gene siRNA injected larvae were separated by 7.5% SDS-PAGE, transferred onto a nitrocellulose membrane, then incubated with biotinylated Cry1Aa toxin (200 ng/mL) followed by further incubation with streptavidin-ALP conjugate (1∶1000 dilutions) and finally developed with NBT-BCIP substrate. Note the substantially reduced interaction of Cry1Aa toxin to the 113 kDa membrane protein of fat body and Malpighian tubule of the target gene knockdown larvae. Lower panels in each blot (β-actin expression) represent equal loading of proteins. Control (Cont): double-stranded GFP siRNA injected insects, Experimental (Expt): double-stranded AjAPN1 siRNA injected insects.

    Journal: PLoS ONE

    Article Title: Functional Interpretation of a Non-Gut Hemocoelic Tissue Aminopeptidase N (APN) in a Lepidopteran Insect Pest Achaea janata

    doi: 10.1371/journal.pone.0079468

    Figure Lengend Snippet: In vitro Cry1Aa toxin interaction in AjAPN1 knockdown larvae. Fat body (Fb), Malpighian tubule (Mt) and salivary gland (Sg) membrane protein fractions (30 µg each) of target and control gene siRNA injected larvae were separated by 7.5% SDS-PAGE, transferred onto a nitrocellulose membrane, then incubated with biotinylated Cry1Aa toxin (200 ng/mL) followed by further incubation with streptavidin-ALP conjugate (1∶1000 dilutions) and finally developed with NBT-BCIP substrate. Note the substantially reduced interaction of Cry1Aa toxin to the 113 kDa membrane protein of fat body and Malpighian tubule of the target gene knockdown larvae. Lower panels in each blot (β-actin expression) represent equal loading of proteins. Control (Cont): double-stranded GFP siRNA injected insects, Experimental (Expt): double-stranded AjAPN1 siRNA injected insects.

    Article Snippet: The pull-down Cry1Aa toxin-interacting proteins were separated by 7.5% SDS-PAGE, electro-blotted onto nitrocellulose membrane (Pall Life Sciences), incubated with A. janata fat body APN polyclonal antibody (1∶10000 dilutions) , followed by incubation with ALP-conjugated goat anti-rabbit IgG and finally developed with NBT-BCIP substrate (Sigma-Aldrich).

    Techniques: In Vitro, Injection, SDS Page, Incubation, ALP Assay, Expressing

    Immunoprecipitation of Cry1Aa toxin interacting protein. Triton X-100 solubilized Malpighian tubule and salivary gland membrane protein fractions (200 µg each) prepared from early fifth instar (5E) larvae were separately incubated with purified activated Cry1Aa toxin (5 µg) followed by further incubation with Cry1Aa polyclonal antibody (2.5 µg). The Cry toxin-interacting protein complex was pull-down with Protein A agarose beads, resolved by 7.5% SDS-PAGE, transferred onto a nitrocellulose membrane, then incubated with A. janata fat body APN polyclonal antibody, followed by incubation with ALP-conjugated secondary antibody and finally developed with NBT-BCIP substrate. Note the detection of a 113 kDa interacting membrane protein in both the tissues. ( − ) and (+) indicate absence and presence of Cry1Aa toxin respectively during incubation.

    Journal: PLoS ONE

    Article Title: Functional Interpretation of a Non-Gut Hemocoelic Tissue Aminopeptidase N (APN) in a Lepidopteran Insect Pest Achaea janata

    doi: 10.1371/journal.pone.0079468

    Figure Lengend Snippet: Immunoprecipitation of Cry1Aa toxin interacting protein. Triton X-100 solubilized Malpighian tubule and salivary gland membrane protein fractions (200 µg each) prepared from early fifth instar (5E) larvae were separately incubated with purified activated Cry1Aa toxin (5 µg) followed by further incubation with Cry1Aa polyclonal antibody (2.5 µg). The Cry toxin-interacting protein complex was pull-down with Protein A agarose beads, resolved by 7.5% SDS-PAGE, transferred onto a nitrocellulose membrane, then incubated with A. janata fat body APN polyclonal antibody, followed by incubation with ALP-conjugated secondary antibody and finally developed with NBT-BCIP substrate. Note the detection of a 113 kDa interacting membrane protein in both the tissues. ( − ) and (+) indicate absence and presence of Cry1Aa toxin respectively during incubation.

    Article Snippet: The pull-down Cry1Aa toxin-interacting proteins were separated by 7.5% SDS-PAGE, electro-blotted onto nitrocellulose membrane (Pall Life Sciences), incubated with A. janata fat body APN polyclonal antibody (1∶10000 dilutions) , followed by incubation with ALP-conjugated goat anti-rabbit IgG and finally developed with NBT-BCIP substrate (Sigma-Aldrich).

    Techniques: Immunoprecipitation, Incubation, Purification, SDS Page, ALP Assay

    The effects of hPTH(1–34) and GR(1–34) on osteogenesis and osteoclastogenesis in bone marrow cells of ORX mice. At the 2nd week of administration, ALP activity of the cells in osteogenic medium were stained and measured with a BCIP/NBT Color Development Kit ( a ); the mineralized nodules were stained with Alizarin Red S solution and calcium deposition was quantified spectrophotometrically ( b ). At the 1st week of administration, TRAP staining was performed and the percentage of TRAP+ cells to total cells were counted ( c ). [Three independent experiments were repeated for ( a b ) and six for ( c ); 10 fields at 100 magnitude were randomly selected for TRAP+ cell counting; * P

    Journal: BMC Musculoskeletal Disorders

    Article Title: Phospholipase C signaling activated by parathyroid hormone mediates the rapid osteoclastogenesis in the fracture healing of orchiectomized mice

    doi: 10.1186/s12891-018-2231-3

    Figure Lengend Snippet: The effects of hPTH(1–34) and GR(1–34) on osteogenesis and osteoclastogenesis in bone marrow cells of ORX mice. At the 2nd week of administration, ALP activity of the cells in osteogenic medium were stained and measured with a BCIP/NBT Color Development Kit ( a ); the mineralized nodules were stained with Alizarin Red S solution and calcium deposition was quantified spectrophotometrically ( b ). At the 1st week of administration, TRAP staining was performed and the percentage of TRAP+ cells to total cells were counted ( c ). [Three independent experiments were repeated for ( a b ) and six for ( c ); 10 fields at 100 magnitude were randomly selected for TRAP+ cell counting; * P

    Article Snippet: The cells were fixed with 4% paraformaldehyde for 30 min, gently rinsed with PBS and stained for ALP with BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime Institute of Biotechnology, Haimen, China), or for mineralized nodule demonstration with 1% Alizarin Red S solution (Sigma, St Louis, MO, USA) for 30 min at room temperature.

    Techniques: Mouse Assay, ALP Assay, Activity Assay, Staining, Cell Counting

    SDS-PAGE and Western blot of rJpIocys2a, rBrBmcys2b and rBrBmcys2c production. Western blot: purified rGST-JpIocys2a probed with anti-GST-Hl primary antibody and rabbit anti-IgG secondary antibody conjugate with alkaline phosphatase; purified rBrBmcys2b and rBrBmcys2c probed with anti-histidine tag primary antibody conjugate with alkaline phosphatase. Alkaline phosphatase revelations were performed with NBT and BCIP. SDS-PAGE: Recombinant cystatins resolved by 14% SDS-PAGE were stained with Coomassie blue G-250; purified rGST-JpIocys2a before and after thrombin cleavage (rGST and JpIocys2a); purified rJpIocys2a, rBrBmcys2b and rBrBmcys2c. MW: molecular weight.

    Journal: Parasites & Vectors

    Article Title: Rhipicephalus microplus and Ixodes ovatus cystatins in tick blood digestion and evasion of host immune response

    doi: 10.1186/s13071-015-0743-3

    Figure Lengend Snippet: SDS-PAGE and Western blot of rJpIocys2a, rBrBmcys2b and rBrBmcys2c production. Western blot: purified rGST-JpIocys2a probed with anti-GST-Hl primary antibody and rabbit anti-IgG secondary antibody conjugate with alkaline phosphatase; purified rBrBmcys2b and rBrBmcys2c probed with anti-histidine tag primary antibody conjugate with alkaline phosphatase. Alkaline phosphatase revelations were performed with NBT and BCIP. SDS-PAGE: Recombinant cystatins resolved by 14% SDS-PAGE were stained with Coomassie blue G-250; purified rGST-JpIocys2a before and after thrombin cleavage (rGST and JpIocys2a); purified rJpIocys2a, rBrBmcys2b and rBrBmcys2c. MW: molecular weight.

    Article Snippet: Alkaline phosphatase revelations were performed with NBT (nitro blue tetrazolium) and BCIP (5-bromo-4-chloro-3-indolyl phosphate, Sigma) in PBS.

    Techniques: SDS Page, Western Blot, Purification, Recombinant, Staining, Molecular Weight

    Cross-immunogenicity between native and recombinant tick cystatins. By Western blot, R. microplus and I. ovatus recombinant cystatins or R. microplus tissue extracts were analyzed using sera (1:50) against: A) rBrBmcys2b; B) rBrBmcys2c C) STQpep. SG, salivary glands; OV, ovary; FB, fatty body, S, saliva; H, hemolymph; L, larva; SGp, salivary glands from partially engorged female; SGt, salivary glands from fully engorged female; C2b, rBrBmcys2b; C2c, rBrBmcys2c; CIo, rJpIocys2a. MW: molecular weight. Anti-IgG alkaline phosphatase rabbit sera and peroxidase hamster sera conjugates were used as secondary antibodies. Alkaline phosphatase revelations were performed with NBT and BCIP. Peroxidase revelations were performed with DAB, H 2 O 2 and CoCl 2 .

    Journal: Parasites & Vectors

    Article Title: Rhipicephalus microplus and Ixodes ovatus cystatins in tick blood digestion and evasion of host immune response

    doi: 10.1186/s13071-015-0743-3

    Figure Lengend Snippet: Cross-immunogenicity between native and recombinant tick cystatins. By Western blot, R. microplus and I. ovatus recombinant cystatins or R. microplus tissue extracts were analyzed using sera (1:50) against: A) rBrBmcys2b; B) rBrBmcys2c C) STQpep. SG, salivary glands; OV, ovary; FB, fatty body, S, saliva; H, hemolymph; L, larva; SGp, salivary glands from partially engorged female; SGt, salivary glands from fully engorged female; C2b, rBrBmcys2b; C2c, rBrBmcys2c; CIo, rJpIocys2a. MW: molecular weight. Anti-IgG alkaline phosphatase rabbit sera and peroxidase hamster sera conjugates were used as secondary antibodies. Alkaline phosphatase revelations were performed with NBT and BCIP. Peroxidase revelations were performed with DAB, H 2 O 2 and CoCl 2 .

    Article Snippet: Alkaline phosphatase revelations were performed with NBT (nitro blue tetrazolium) and BCIP (5-bromo-4-chloro-3-indolyl phosphate, Sigma) in PBS.

    Techniques: Recombinant, Western Blot, Molecular Weight

    (A) Phenotypes of colonies from F. oxysporum wild-type and mutant strains, showing alterations in cell wall or membrane integrity. (B) Germlings from the wild-type (wt) strain and Δ chsV , Δ chsVb , and Δ chsV Δ chsVb mutants grown on SM with or without (w/o) 1.2 M sorbitol (magnification, ×50). Arrows indicate swollen ballon-like structures. BCIP, 5-bromo-4-chloro-3-indolylphosphate; SDS, sodium dodecyl sulfate; CR, Congo red; CFW, calcofluor white.

    Journal: Eukaryotic Cell

    Article Title: ChsVb, a Class VII Chitin Synthase Involved in Septation, Is Critical for Pathogenicity in Fusarium oxysporum ▿ ▿ †

    doi: 10.1128/EC.00347-07

    Figure Lengend Snippet: (A) Phenotypes of colonies from F. oxysporum wild-type and mutant strains, showing alterations in cell wall or membrane integrity. (B) Germlings from the wild-type (wt) strain and Δ chsV , Δ chsVb , and Δ chsV Δ chsVb mutants grown on SM with or without (w/o) 1.2 M sorbitol (magnification, ×50). Arrows indicate swollen ballon-like structures. BCIP, 5-bromo-4-chloro-3-indolylphosphate; SDS, sodium dodecyl sulfate; CR, Congo red; CFW, calcofluor white.

    Article Snippet: When needed, SM was supplemented with 5-bromo-4-chloro-3-indolylphosphate (BCIP), Congo red, calcofluor white (CFW), sodium dodecyl sulfate (SDS), and menadione at the indicated concentrations (all from Sigma-Aldrich Química, Spain).

    Techniques: Mutagenesis

    Histone H2AX phosphorylated at S139 (H2AXS139Ph) is a marker of double-stranded breaks (DSBs), while poly(ADP-ribose) polymerase-2 (PARP-2) is a marker of single-stranded breaks (SSBs). (A) Results of the immunocytochemical analysis and the method of tissue printing . (a-a', b-c) the presentation of superimposed fluorescence images (DAPI-related in blue and H2AXS139Ph-related in green) after the immunocytochemical detection of H2AXS139Ph in (a) control, (b) after 2.5 mM hydroxyurea-treatment (HU) for 32 h, (c) after 24-h synchronization under the influence of 2.5 mM HU and 8-h co-treatment with 2.5 mM HU and 5 mM caffeine (CF). (a') negative control; incubation exclusively with secondary antibodies. The values of marking indices (expressed in percents) are presented in the top left corner on the following images: (a)–for control series; (b)–after 32-h treatment with HU; (c)–after the induction of premature chromosome condensation (PCC) under the influence of HU/CF. Scale bars in a-a', b-c are 20 μm. (d-f) identification of H2AXS139Ph in the top sections of Vicia faba roots by the method of tissue printing , negative images. In the top left corner of each negative image, there is a miniature of the same fragment of nitrocellulose membrane in color, i.e. stained in the reaction of NBT/BCIP (d'-f'). (d-d') control, (e-e') HU, 32 h, (f-f') HU for 24 h and co-incubation HU/CF for 8 h (total incubation time: 32 h). Scale bars in d'-f' and d-f are 10 mm. (g-g', h-i) presentation of superimposed fluorescence images (DAPI-related in blue and PARP-2-related in green) after the immunocytochemical detection of PARP-2: (g) control, (h) after HU-treatment for 32 h, (i) after 24-h synchronization under the influence HU and 8-h co-treatment with HU/CF. (g') negative control; incubation exclusively with secondary antibodies. The values of marking (expressed in percents) are presented in the top left corner of the following images (g) control series; (h) after 32-h treatment with HU; (i) after the induction of PCC under the influence of HU/CF. Scale bars in g-g', h-i are 20 μm. (j-l) identification of PARP-2 in the top section of V . faba roots by the method of tissue printing , negative images. In the top left corner of each negative image, there is a miniature of the same fragment of nitrocellulose membrane in color, i.e. stained in the reaction of NBT/BCIP (j'-l'). (j-j') control, (k-k') HU, 32 h, (l-l') HU for 24 h and co-incubation HU/CF. Scale bars in j'-l' and j-l are 10 mm. (B) Identification of proteins H2AXS139Ph and PARP-2 by the method of Western blot. (a-a') expression levels of the H2AXS139Ph by Western blot analysis. Data shown are the representatives of three independent experiments. The relative levels of H2AXS139Ph after normalization for actin, as determined by densitometry analysis of the bands, are shown in the histogram (a'; the pixel values [ pv ; 1–255] categorized according to densitometry analysis of the band intensities and expressed in arbitrary units [a.u.]). Columns , mean from three independent experiments; bars , SD. * p ≤ 0.001 (Control/HU, Mann-Whitney U test); ▲ p ≤ 0.01 (Control/PCC, Mann-Whitney U test). (b-b') expression levels of the PARP-2 by Western blot analysis. Data shown are representative of three independent experiments. The relative levels of PARP-2 after normalization for actin, as determined by densitometry analysis of the bands, are shown in the histogram (b'; the pixel values [ pv ; 1–255] categorized according to densitometry analysis of the band intensities and expressed in arbitrary units [a.u.]). Columns , mean from three independent experiments; bars , SD. ▲ p ≤ 0.01 (Control/HU, Mann-Whitney U test); * p ≤ 0.001 (Control/PCC, Mann-Whitney U test).

    Journal: PLoS ONE

    Article Title: Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba

    doi: 10.1371/journal.pone.0142307

    Figure Lengend Snippet: Histone H2AX phosphorylated at S139 (H2AXS139Ph) is a marker of double-stranded breaks (DSBs), while poly(ADP-ribose) polymerase-2 (PARP-2) is a marker of single-stranded breaks (SSBs). (A) Results of the immunocytochemical analysis and the method of tissue printing . (a-a', b-c) the presentation of superimposed fluorescence images (DAPI-related in blue and H2AXS139Ph-related in green) after the immunocytochemical detection of H2AXS139Ph in (a) control, (b) after 2.5 mM hydroxyurea-treatment (HU) for 32 h, (c) after 24-h synchronization under the influence of 2.5 mM HU and 8-h co-treatment with 2.5 mM HU and 5 mM caffeine (CF). (a') negative control; incubation exclusively with secondary antibodies. The values of marking indices (expressed in percents) are presented in the top left corner on the following images: (a)–for control series; (b)–after 32-h treatment with HU; (c)–after the induction of premature chromosome condensation (PCC) under the influence of HU/CF. Scale bars in a-a', b-c are 20 μm. (d-f) identification of H2AXS139Ph in the top sections of Vicia faba roots by the method of tissue printing , negative images. In the top left corner of each negative image, there is a miniature of the same fragment of nitrocellulose membrane in color, i.e. stained in the reaction of NBT/BCIP (d'-f'). (d-d') control, (e-e') HU, 32 h, (f-f') HU for 24 h and co-incubation HU/CF for 8 h (total incubation time: 32 h). Scale bars in d'-f' and d-f are 10 mm. (g-g', h-i) presentation of superimposed fluorescence images (DAPI-related in blue and PARP-2-related in green) after the immunocytochemical detection of PARP-2: (g) control, (h) after HU-treatment for 32 h, (i) after 24-h synchronization under the influence HU and 8-h co-treatment with HU/CF. (g') negative control; incubation exclusively with secondary antibodies. The values of marking (expressed in percents) are presented in the top left corner of the following images (g) control series; (h) after 32-h treatment with HU; (i) after the induction of PCC under the influence of HU/CF. Scale bars in g-g', h-i are 20 μm. (j-l) identification of PARP-2 in the top section of V . faba roots by the method of tissue printing , negative images. In the top left corner of each negative image, there is a miniature of the same fragment of nitrocellulose membrane in color, i.e. stained in the reaction of NBT/BCIP (j'-l'). (j-j') control, (k-k') HU, 32 h, (l-l') HU for 24 h and co-incubation HU/CF. Scale bars in j'-l' and j-l are 10 mm. (B) Identification of proteins H2AXS139Ph and PARP-2 by the method of Western blot. (a-a') expression levels of the H2AXS139Ph by Western blot analysis. Data shown are the representatives of three independent experiments. The relative levels of H2AXS139Ph after normalization for actin, as determined by densitometry analysis of the bands, are shown in the histogram (a'; the pixel values [ pv ; 1–255] categorized according to densitometry analysis of the band intensities and expressed in arbitrary units [a.u.]). Columns , mean from three independent experiments; bars , SD. * p ≤ 0.001 (Control/HU, Mann-Whitney U test); ▲ p ≤ 0.01 (Control/PCC, Mann-Whitney U test). (b-b') expression levels of the PARP-2 by Western blot analysis. Data shown are representative of three independent experiments. The relative levels of PARP-2 after normalization for actin, as determined by densitometry analysis of the bands, are shown in the histogram (b'; the pixel values [ pv ; 1–255] categorized according to densitometry analysis of the band intensities and expressed in arbitrary units [a.u.]). Columns , mean from three independent experiments; bars , SD. ▲ p ≤ 0.01 (Control/HU, Mann-Whitney U test); * p ≤ 0.001 (Control/PCC, Mann-Whitney U test).

    Article Snippet: Signals were visualized with NBT/BCIP (Nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate, toluidine salt, Sigma-Aldrich, Saint Quentin, France) as substrates.

    Techniques: Marker, Fluorescence, Negative Control, Incubation, Periodic Counter-current Chromatography, Staining, Western Blot, Expressing, MANN-WHITNEY

    Immunoprecipitation and Western-blot analysis of DEL, KMH2 and L-428 cell lysates. 23.1 (Panel A and C) and R24.1 (Panel B and D). Samples were separated on 4–20% Life-gel and transferred onto nitrocellulose membranes. The membranes were then blocked in 5% non-fat dry milk and probed with monoclonal antibody clones R23.1 (Panel A and B) and R24.1 (Panel C and D) followed by goat-anti-mouse IgG-AP. The membranes were developed in BCIP/NBT. A protein band of ~21 KDa as well as IgG light chain were detected in all three lysates examined. Lane 1 contains molecular weights and lanes 2–4 contain Immunoprecipitated samples of DEL, KMH2 and L-428.

    Journal: Molecular Cancer

    Article Title: Constitutive overexpression of a novel 21 kDa protein by Hodgkin Lymphoma and Aggressive Non-Hodgkin Lymphomas

    doi: 10.1186/1476-4598-7-12

    Figure Lengend Snippet: Immunoprecipitation and Western-blot analysis of DEL, KMH2 and L-428 cell lysates. 23.1 (Panel A and C) and R24.1 (Panel B and D). Samples were separated on 4–20% Life-gel and transferred onto nitrocellulose membranes. The membranes were then blocked in 5% non-fat dry milk and probed with monoclonal antibody clones R23.1 (Panel A and B) and R24.1 (Panel C and D) followed by goat-anti-mouse IgG-AP. The membranes were developed in BCIP/NBT. A protein band of ~21 KDa as well as IgG light chain were detected in all three lysates examined. Lane 1 contains molecular weights and lanes 2–4 contain Immunoprecipitated samples of DEL, KMH2 and L-428.

    Article Snippet: The protein bands were finally visualized in BCIP/NBT substrate (Sigma, MO, USA).

    Techniques: Immunoprecipitation, Western Blot, Clone Assay

    Titers and cross-reactivity of antivenoms raised against B . arietans and Bitis g . rhinoceros plus B . nasicornis venoms. [A] ELISA : ELISA plates were coated with 1.0 μg of the Bitis spp venoms/well and incubated with different dilutions of the horse experimental antivenoms produced against B . arietans venom (α-Ba antivenom), B . g . rhinoceros plus B . nasicornis venoms (α-Br + Bn antivenom) or with the botulinic toxin antiserum (control), followed by anti-horse IgG-HRPO-conjugated. The results were expressed as the mean of absorbance value ± SD. [B] Western Blot : venom samples (5 μg) from B . arietans (Ba), B . g . rhinoceros (Br) and B . nasicornis (Bn) snakes were separated by SDS-PAGE, electrotransfered to nitrocellulose membranes and incubated with the antivenoms raised against B . arietans (α-Ba), B . g . rhinoceros plus B . nasicornis (α-Br+Bn) or with botulinic toxin antiserum (control) diluted 1:2000 followed by GAH/Ig-AP. The reactions were revealed with NBT and BCIP.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: African Adders: Partial Characterization of Snake Venoms from Three Bitis Species of Medical Importance and Their Neutralization by Experimental Equine Antivenoms

    doi: 10.1371/journal.pntd.0003419

    Figure Lengend Snippet: Titers and cross-reactivity of antivenoms raised against B . arietans and Bitis g . rhinoceros plus B . nasicornis venoms. [A] ELISA : ELISA plates were coated with 1.0 μg of the Bitis spp venoms/well and incubated with different dilutions of the horse experimental antivenoms produced against B . arietans venom (α-Ba antivenom), B . g . rhinoceros plus B . nasicornis venoms (α-Br + Bn antivenom) or with the botulinic toxin antiserum (control), followed by anti-horse IgG-HRPO-conjugated. The results were expressed as the mean of absorbance value ± SD. [B] Western Blot : venom samples (5 μg) from B . arietans (Ba), B . g . rhinoceros (Br) and B . nasicornis (Bn) snakes were separated by SDS-PAGE, electrotransfered to nitrocellulose membranes and incubated with the antivenoms raised against B . arietans (α-Ba), B . g . rhinoceros plus B . nasicornis (α-Br+Bn) or with botulinic toxin antiserum (control) diluted 1:2000 followed by GAH/Ig-AP. The reactions were revealed with NBT and BCIP.

    Article Snippet: Immunoreactive proteins were detected using NBT/BCIP according to the manufacturer’s instructions (Promega).

    Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Produced, Western Blot, SDS Page

    The distribution of miR-101 in breast tissues. The location of miR-101 in breast cancer tissues and adjacent normal breast tissues was subjected to in situ hybridization using DIG-labeled LNA probes specific to miR-101. The sections were hybridized with DIG-labeled LNA scrambled miRNA probe as a negative control ( Figure S1A ). The stain was developed with BCIP/NBT. Black arrows indicate hybridization signal. The scale bar indicated a distance of 500 µm.

    Journal: PLoS ONE

    Article Title: MiR-101 Is Involved in Human Breast Carcinogenesis by Targeting Stathmin1

    doi: 10.1371/journal.pone.0046173

    Figure Lengend Snippet: The distribution of miR-101 in breast tissues. The location of miR-101 in breast cancer tissues and adjacent normal breast tissues was subjected to in situ hybridization using DIG-labeled LNA probes specific to miR-101. The sections were hybridized with DIG-labeled LNA scrambled miRNA probe as a negative control ( Figure S1A ). The stain was developed with BCIP/NBT. Black arrows indicate hybridization signal. The scale bar indicated a distance of 500 µm.

    Article Snippet: The sections were then incubated in buffer containing anti-DIG-antibody (Roche, Mannheim, Germany) 2 h at 37°C, followed by staining with NBT and BCIP (Promega, Madison, WI, USA).

    Techniques: In Situ Hybridization, Labeling, Negative Control, Staining, Hybridization

    Differentiation phenotypes in BRM- and BRG1-depleted cells. A , the multisubunit SWI/SNF chromatin-remodeling complex contains a core ATPase, either BRG1 or BRM, plus seven or more non-catalytic subunits. Of these, ARID1A and ARID1B are also mutually exclusive alternatives. Either ATPase can associate with either ARID family member ( 25 , 35 ), such that there are at least four distinct subsets of the SWI/SNF complex. B , parental and knockdown cell cultures were induced for the time intervals indicated, fixed with methanol, and reacted with the substrate BCIP/NBT to reveal alkaline phosphatase activity; positive cells stain purple-black. Seq , sequence. C , induced cell monolayers were stained at later time intervals with Alizarin Red S, which indicates the presence of calcium-containing compounds in the cell matrix. D , the phenotype of three independent BRM knockdown lines, generated with two different shRNA sequences, was analyzed as described in panels B and C. E , total cell lysate from parental and knockdown lines (indicated above the lanes) was probed with antibodies specific to either BRM or BRG1, as indicated to the right . An antibody probe for the constitutively expressed HSC70 protein was used as a loading control.

    Journal: The Journal of Biological Chemistry

    Article Title: Antagonistic Roles for BRM and BRG1 SWI/SNF Complexes in Differentiation *Antagonistic Roles for BRM and BRG1 SWI/SNF Complexes in Differentiation * ♦

    doi: 10.1074/jbc.M808782200

    Figure Lengend Snippet: Differentiation phenotypes in BRM- and BRG1-depleted cells. A , the multisubunit SWI/SNF chromatin-remodeling complex contains a core ATPase, either BRG1 or BRM, plus seven or more non-catalytic subunits. Of these, ARID1A and ARID1B are also mutually exclusive alternatives. Either ATPase can associate with either ARID family member ( 25 , 35 ), such that there are at least four distinct subsets of the SWI/SNF complex. B , parental and knockdown cell cultures were induced for the time intervals indicated, fixed with methanol, and reacted with the substrate BCIP/NBT to reveal alkaline phosphatase activity; positive cells stain purple-black. Seq , sequence. C , induced cell monolayers were stained at later time intervals with Alizarin Red S, which indicates the presence of calcium-containing compounds in the cell matrix. D , the phenotype of three independent BRM knockdown lines, generated with two different shRNA sequences, was analyzed as described in panels B and C. E , total cell lysate from parental and knockdown lines (indicated above the lanes) was probed with antibodies specific to either BRM or BRG1, as indicated to the right . An antibody probe for the constitutively expressed HSC70 protein was used as a loading control.

    Article Snippet: Proteins were separated by polyacrylamide gel electrophoresis, transferred to Immobilon-P membrane (Millipore), and visualized using either Western Lighting chemiluminescence reagent Plus (PerkinElmer Life Sciences) or BCIP/NBT (Promega).

    Techniques: Activity Assay, Staining, Sequencing, Generated, shRNA

    Continuous exposure to gemcitabine increases tumorigenicity but continuous exposure to sulforaphane or quercetin reduces it. ( a ) BxPC-3, Bx-GEM, Bx-Q and Bx-SF cells were seeded at a low density (2000 cells/well) in 6-well plates. After 2 weeks, cells were Coomassie-stained and colonies containing more than 50 cells were counted under a dissecting microscope. The survival fraction and representative photographs of colonies (first generation) are presented on the left. For second-generation colony formation, an equal amount of living cells from first-generation colonies were collected and 2000 cells per well were re-seeded. The colony formation was analyzed as described above and is presented on the right. ( b ) Cells were cultured to 90% confluence before the cell layer was scratched with the tip of a 10- μ l pipette. Closure of the wounded region was evaluated 24 h after scratching by microscopy at × 100 magnification. For quantification of the scratched area, the percentage of the gap area was evaluated and calculated by TScratch software (diagram below photographs). ( c ) Cells were seeded in 6-well plates, followed by exposure to NH Osteo-Diff medium to induce osteocytic differentiation. Fourteen days later, the cells were stained with BCIP/NBT substrate for alkaline phosphatases, expressed by cells differentiated into osteocytes, which appear dark. Representative images at × 200 magnification are shown. ( d ) Proteins were harvested and the expression of EpCAM, Nanog, Twist2 and E-cadherin was measured by western blot analysis. β -Actin was used as a loading control. Three independent experiments were performed at least in triplicates and the data are presented as means ±S.D. * P

    Journal: Cell Death & Disease

    Article Title: Continuous exposure of pancreatic cancer cells to dietary bioactive agents does not induce drug resistance unlike chemotherapy

    doi: 10.1038/cddis.2016.157

    Figure Lengend Snippet: Continuous exposure to gemcitabine increases tumorigenicity but continuous exposure to sulforaphane or quercetin reduces it. ( a ) BxPC-3, Bx-GEM, Bx-Q and Bx-SF cells were seeded at a low density (2000 cells/well) in 6-well plates. After 2 weeks, cells were Coomassie-stained and colonies containing more than 50 cells were counted under a dissecting microscope. The survival fraction and representative photographs of colonies (first generation) are presented on the left. For second-generation colony formation, an equal amount of living cells from first-generation colonies were collected and 2000 cells per well were re-seeded. The colony formation was analyzed as described above and is presented on the right. ( b ) Cells were cultured to 90% confluence before the cell layer was scratched with the tip of a 10- μ l pipette. Closure of the wounded region was evaluated 24 h after scratching by microscopy at × 100 magnification. For quantification of the scratched area, the percentage of the gap area was evaluated and calculated by TScratch software (diagram below photographs). ( c ) Cells were seeded in 6-well plates, followed by exposure to NH Osteo-Diff medium to induce osteocytic differentiation. Fourteen days later, the cells were stained with BCIP/NBT substrate for alkaline phosphatases, expressed by cells differentiated into osteocytes, which appear dark. Representative images at × 200 magnification are shown. ( d ) Proteins were harvested and the expression of EpCAM, Nanog, Twist2 and E-cadherin was measured by western blot analysis. β -Actin was used as a loading control. Three independent experiments were performed at least in triplicates and the data are presented as means ±S.D. * P

    Article Snippet: Then, the differentiation potential was tested by culturing the cells in osteogenic differentiation medium for 14 days, followed by staining with SIGMAFAST BCIP/NBT (Sigma-Aldrich, St. Louis, MO, USA) substrate for detection of alkaline phosphatase produced by cells differentiated into osteoblasts.

    Techniques: Staining, Microscopy, Cell Culture, Transferring, Software, Expressing, Western Blot

    Dot blots showing binding of biotinylated fibrinogen by S. iniae . Strains QMA0076 and QMA0141 were incubated with biotinylated fibrinogen, washed and transferred to PVDF, then membranes were probed with streptavidin-alkaline phosphatase conjugate and developed with NBT/BCIP. Panel A) Lanes 1 and 2 comprised 5 μl cells grown in vegetable peptone, whilst lanes 2 and 3 contained 5 μl cells grown in barramundi serum. Cells were harvested and washed in TBS before being incubated with biotinylated human fibrinogen (Lanes 1 and 3) or TBS (lanes 2 and 4) for 20 min, prior to extensive washing and transfer onto PVDF membrane for subsequent detection. Fb indicates serial two-fold dilutions of biotinylated fibrinogen commencing at 2.5 μg/mL. Panel B) shows serial 2 × dilutions of 5 μl cells diluted in TBS. Lane F, cells incubated first with TBS then with biotinylated fibrinogen before transfer to the membrane. Lane P+F, cells incubated with barramundi plasma followed by biotinylated fibrinogen, Lane TBS, cells incubated twice in TBS.

    Journal: BMC Microbiology

    Article Title: Identification and molecular characterisation of a fibrinogen binding protein from Streptococcus iniae.

    doi: 10.1186/1471-2180-8-67

    Figure Lengend Snippet: Dot blots showing binding of biotinylated fibrinogen by S. iniae . Strains QMA0076 and QMA0141 were incubated with biotinylated fibrinogen, washed and transferred to PVDF, then membranes were probed with streptavidin-alkaline phosphatase conjugate and developed with NBT/BCIP. Panel A) Lanes 1 and 2 comprised 5 μl cells grown in vegetable peptone, whilst lanes 2 and 3 contained 5 μl cells grown in barramundi serum. Cells were harvested and washed in TBS before being incubated with biotinylated human fibrinogen (Lanes 1 and 3) or TBS (lanes 2 and 4) for 20 min, prior to extensive washing and transfer onto PVDF membrane for subsequent detection. Fb indicates serial two-fold dilutions of biotinylated fibrinogen commencing at 2.5 μg/mL. Panel B) shows serial 2 × dilutions of 5 μl cells diluted in TBS. Lane F, cells incubated first with TBS then with biotinylated fibrinogen before transfer to the membrane. Lane P+F, cells incubated with barramundi plasma followed by biotinylated fibrinogen, Lane TBS, cells incubated twice in TBS.

    Article Snippet: Resolved IPTG-induced cellular lysates were blotted onto PVDF membranes using a semi dry apparatus (Hoeffer Semi-Phor, GE Healthcare, North Ryde, NSW), the membranes were probed with biotinylated fibrinogen (5 μg/mL) prepared as described above and detected using streptavidin-alkaline phosphatase (Pierce, Rockford, Il.) followed by colour development using 1-Step NBT-BCIP (Pierce, Rockford Il.).

    Techniques: Binding Assay, Incubation

    Establishment of transgenic cell lines stably expressing EGFP and mCherry (RFP). (a) A schematic illustration of the constructed ΔDE-pOG2-ΔPE-pOm2 vector using the porcine Oct4 promoter. DE: distal enhancer; PE: proximal enhancer. (b) Genotyping the Oct4 promoter-driven transgenes from genomic DNA. PC: positive control; DW: distilled water, negative control; WT: wild type; TG: transgenic cell lines. Lanes 3–6, fluorescence-positive cell lines established from wild type F9 cells, P19 cells, mESCs, and MEFs. (c) Reverse transcriptional PCR analysis of pluripotency markers in fluorescence-positive cell lines. (d) Surface alkaline phosphatase activity in transgenic F9 cells, P19 cells, and mESCs was measured using BCIP/NBT. (e) Typical pluripotency markers, Nanog and Oct4, were assayed by immunofluorescent cytochemical staining in transgenic F9 cells, P19 cells, and mESCs. Scale bars indicate 20 μ m.

    Journal: Stem Cells International

    Article Title: Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation

    doi: 10.1155/2016/1390284

    Figure Lengend Snippet: Establishment of transgenic cell lines stably expressing EGFP and mCherry (RFP). (a) A schematic illustration of the constructed ΔDE-pOG2-ΔPE-pOm2 vector using the porcine Oct4 promoter. DE: distal enhancer; PE: proximal enhancer. (b) Genotyping the Oct4 promoter-driven transgenes from genomic DNA. PC: positive control; DW: distilled water, negative control; WT: wild type; TG: transgenic cell lines. Lanes 3–6, fluorescence-positive cell lines established from wild type F9 cells, P19 cells, mESCs, and MEFs. (c) Reverse transcriptional PCR analysis of pluripotency markers in fluorescence-positive cell lines. (d) Surface alkaline phosphatase activity in transgenic F9 cells, P19 cells, and mESCs was measured using BCIP/NBT. (e) Typical pluripotency markers, Nanog and Oct4, were assayed by immunofluorescent cytochemical staining in transgenic F9 cells, P19 cells, and mESCs. Scale bars indicate 20 μ m.

    Article Snippet: Alkaline Phosphatase (AP) Staining Alkaline phosphatase (AP) staining was performed according to a previously reported procedure [ ] using the BCIP®/NBT Liquid Substrate System (Sigma, USA).

    Techniques: Transgenic Assay, Stable Transfection, Expressing, Construct, Plasmid Preparation, Positive Control, Negative Control, Fluorescence, Polymerase Chain Reaction, Activity Assay, Staining

    MiR-17-5p and miR-27b-3p were differentially expressed during osteogenic differentiation of human ligament fibroblasts ( A ) ALP in fibroblasts was stained using the BCIP/NBT kit after the cells were incubated in OM for 7 and 14 days. Scale bar = 200 μm. ( B ) The ALP activity of fibroblasts was measured after they were incubated in OM for 7 and 14 days. ( C ) Fibroblasts were incubated in OM for 21 days, and then the mineralized nodules were stained using alizarin red S (ARS). Scale bar = 200 μm. ( D ) Mineralization was quantified by extraction of ARS dye with 10% cetylpyridinium chloride. ( E ) The total RNA was isolated on days 7 and 14. Runx2, ALP, and COL1A1 mRNA levels were determined using qRT-PCR and normalized to GAPDH. ( F ) The total RNA was isolated on days 7 and 14. Levels of miR-17-5p and miR-27b-3p were determined using qRT-PCR and normalized to U6. * P

    Journal: Oncotarget

    Article Title: Differentially expressed mRNAs, lncRNAs, and miRNAs with associated co-expression and ceRNA networks in ankylosing spondylitis

    doi: 10.18632/oncotarget.22708

    Figure Lengend Snippet: MiR-17-5p and miR-27b-3p were differentially expressed during osteogenic differentiation of human ligament fibroblasts ( A ) ALP in fibroblasts was stained using the BCIP/NBT kit after the cells were incubated in OM for 7 and 14 days. Scale bar = 200 μm. ( B ) The ALP activity of fibroblasts was measured after they were incubated in OM for 7 and 14 days. ( C ) Fibroblasts were incubated in OM for 21 days, and then the mineralized nodules were stained using alizarin red S (ARS). Scale bar = 200 μm. ( D ) Mineralization was quantified by extraction of ARS dye with 10% cetylpyridinium chloride. ( E ) The total RNA was isolated on days 7 and 14. Runx2, ALP, and COL1A1 mRNA levels were determined using qRT-PCR and normalized to GAPDH. ( F ) The total RNA was isolated on days 7 and 14. Levels of miR-17-5p and miR-27b-3p were determined using qRT-PCR and normalized to U6. * P

    Article Snippet: The ALP staining assay was performed using a BCIP/NBT ALP Color Development Kit (Beyotime, Shanghai, China).

    Techniques: ALP Assay, Staining, Incubation, Activity Assay, Isolation, Quantitative RT-PCR