Binding Assay:Article Title: IOA-244, a novel p110δ PI3K inhibitor, blocks breast tumour progression on either mono- or combined-therapy.
Article Snippet: .. Sections were then blocked to prevent nonspecific binding using goat serum (for pAkt staining) or 1% BSA for 1 hour at room temperature (for the other antibodies), and subsequently incubated with primary antibodies overnight at 4 °C (pAkt, 1:50; Cell Signaling #4060; F4/80, 1:50; BioRad #MCA497GA; vimentin, 1:200; Thermo Scientific #RM-9120). .. After washing, sections were incubated with HRP-conjugated anti-rat secondary antibody (for F4/80) or HRP-conjugated antirabbit secondary antibody (for pAkt and vimentin), developed using DAB and H2O2, counterstained with hematoxylin, and mounted with Vectashield mounting medium (Vector Laboratories).
Staining:Article Title: IOA-244, a novel p110δ PI3K inhibitor, blocks breast tumour progression on either mono- or combined-therapy.
Article Snippet: .. Sections were then blocked to prevent nonspecific binding using goat serum (for pAkt staining) or 1% BSA for 1 hour at room temperature (for the other antibodies), and subsequently incubated with primary antibodies overnight at 4 °C (pAkt, 1:50; Cell Signaling #4060; F4/80, 1:50; BioRad #MCA497GA; vimentin, 1:200; Thermo Scientific #RM-9120). .. After washing, sections were incubated with HRP-conjugated anti-rat secondary antibody (for F4/80) or HRP-conjugated antirabbit secondary antibody (for pAkt and vimentin), developed using DAB and H2O2, counterstained with hematoxylin, and mounted with Vectashield mounting medium (Vector Laboratories).
Incubation:Article Title: IOA-244, a novel p110δ PI3K inhibitor, blocks breast tumour progression on either mono- or combined-therapy.
Article Snippet: .. Sections were then blocked to prevent nonspecific binding using goat serum (for pAkt staining) or 1% BSA for 1 hour at room temperature (for the other antibodies), and subsequently incubated with primary antibodies overnight at 4 °C (pAkt, 1:50; Cell Signaling #4060; F4/80, 1:50; BioRad #MCA497GA; vimentin, 1:200; Thermo Scientific #RM-9120). .. After washing, sections were incubated with HRP-conjugated anti-rat secondary antibody (for F4/80) or HRP-conjugated antirabbit secondary antibody (for pAkt and vimentin), developed using DAB and H2O2, counterstained with hematoxylin, and mounted with Vectashield mounting medium (Vector Laboratories).
Article Title: Cytoskeletal remodeling promotes tunneling nanotube formation and drives cardiac resident cell mitochondrial transfer in sepsis.
Article Snippet: Paraffin- embedded heart sections (5 μm) were deparaffinized, rehydrated through an ethanol gradient, and subjected to antigen retrieval using sodium citrate buffer (Servicebio, China). .. After blocking with goat serum, sections were incubated overnight at 4°C with primary antibodies [Cldn5 (1:200; Cell Signaling, #66879), Metal (:200; Abcam, ab12228), Cxcl2 (1:200; Abcam, ab317569), Gsn (1:200; Proteintech, 11644- 2- AP), Fmo2 (1:200; Proteintech, 67019- 1- Ig), Lcn2 (1:200; Proteintech, 26991- 1- AP), CD74 (1:200; Abcam, ab289885), troponin (1:200; Abcam, ab209813), CD31 (1:200; Abcam, ab182981), PDGFR (1:200; Abcam, ab203491), CD68 (1:200; Abcam, ab283654), and actin (1:200; Abcam, ab8227)]. .. The following day, horseradish peroxidase polymer–labeled secondary antibodies (1:200; Abcam, ab6721) were applied, followed by tyramide signal amplification (TSA) with TGseries tyramide fluorophores (TG 520N, TG 570N, TG 620N, TG 760N, and TG 700N) for signal amplification.
Article Title: Cytoskeletal remodeling promotes tunneling nanotube formation and drives cardiac resident cell mitochondrial transfer in sepsis
Article Snippet: Paraffin-embedded heart sections (5 μm) were deparaffinized, rehydrated through an ethanol gradient, and subjected to antigen retrieval using sodium citrate buffer (Servicebio, China). .. After blocking with goat serum, sections were incubated overnight at 4°C with primary antibodies [Cldn5 (1:200; Cell Signaling, #66879), Metal (:200; Abcam, ab12228), Cxcl2 (1:200; Abcam, ab317569), Gsn (1:200; Proteintech, 11644-2-AP), Fmo2 (1:200; Proteintech, 67019-1-Ig), Lcn2 (1:200; Proteintech, 26991-1-AP), CD74 (1:200; Abcam, ab289885), troponin (1:200; Abcam, ab209813), CD31 (1:200; Abcam, ab182981), PDGFR (1:200; Abcam, ab203491), CD68 (1:200; Abcam, ab283654), and actin (1:200; Abcam, ab8227)]. .. The following day, horseradish peroxidase polymer–labeled secondary antibodies (1:200; Abcam, ab6721) were applied, followed by tyramide signal amplification (TSA) with TG-series tyramide fluorophores (TG 520N, TG 570N, TG 620N, TG 760N, and TG 700N) for signal amplification.
Article Title: MAP2K6 directly phosphorylates BCL2L13 to mediate mitophagy for suppressing tumorigenicity.
Article Snippet: IHC was performed according to the Immunohistochemical secondary antibody kit instructions (Absin Bioscience Inc., abs996). .. The slides were incubated in 3% hydrogen peroxide and then preincubated with 5% goat serum (Cell Signaling, 5425S). .. Anti-MKK3 (Abcam, ab195037, 1:200), anti-MKK6 (Proteintech, 12745-1-AP, 1:200) and anti-Ki67 (Cell Signaling, 9449, 1:200) were incubated with the slides overnight at 4◦C.
Article Title: Cyclophilin D, Regulator of Mitochondrial Permeability Transition and Bioenergetics, Promotes Adipogenic Differentiation of Mesenchymal Stem Cells
Article Snippet: .. Then sections were washed with 1× PBS and blocked with 2.5% goat serum for 30 min and incubated with perilipin-1 primary antibody (Cell Signaling Technology, Danvers, MA, USA, #9349) at 1:100 dilution in PBS + 0.1% Tween-20 and 5% normal goat serum at room temperature for 2 h. Sections were then incubated with anti-rabbit IgG secondary antibody conjugated with Alexa Fluor ® 647 (Jackson ImmunoResearch, West Grove, PA, USA, RRID: AB_2338072) at 1:200 dilution for 1 h. Fluoroshield Mounting Medium with DAPI (Abcam, Cambridge, UK, ab104139) was used for counterstaining. ..
Immunofluorescence:Article Title: c-Myc transactivates CFL1 to induce senescence-like phenotype and potentiate the bystander effects for the migration and proliferation in lung cancer cells.
Article Snippet: .. The slides were blocked using the immunofluorescence blocking buffer containing goat serum as a protein blocker (Cat#12411, Cell Signaling Technology, Danvers, MA, USA) at 37°C for 45 min. .. The primary antibody, including anti-cofilin-1 (GTX102156) and anti-c-Myc (GTX20032, Genetex Inc., Irvine, CA, USA) were then used to treat cells at 37°C for 1.5 h. AR TI CL E IN P RE SS Subsequently, the slides were rinsed with pre-warmed washing buffer in jar at 45°C for 10 min three times on a shaker.
Blocking Assay:Article Title: c-Myc transactivates CFL1 to induce senescence-like phenotype and potentiate the bystander effects for the migration and proliferation in lung cancer cells.
Article Snippet: .. The slides were blocked using the immunofluorescence blocking buffer containing goat serum as a protein blocker (Cat#12411, Cell Signaling Technology, Danvers, MA, USA) at 37°C for 45 min. .. The primary antibody, including anti-cofilin-1 (GTX102156) and anti-c-Myc (GTX20032, Genetex Inc., Irvine, CA, USA) were then used to treat cells at 37°C for 1.5 h. AR TI CL E IN P RE SS Subsequently, the slides were rinsed with pre-warmed washing buffer in jar at 45°C for 10 min three times on a shaker.
Article Title: Cytoskeletal remodeling promotes tunneling nanotube formation and drives cardiac resident cell mitochondrial transfer in sepsis.
Article Snippet: Paraffin- embedded heart sections (5 μm) were deparaffinized, rehydrated through an ethanol gradient, and subjected to antigen retrieval using sodium citrate buffer (Servicebio, China). .. After blocking with goat serum, sections were incubated overnight at 4°C with primary antibodies [Cldn5 (1:200; Cell Signaling, #66879), Metal (:200; Abcam, ab12228), Cxcl2 (1:200; Abcam, ab317569), Gsn (1:200; Proteintech, 11644- 2- AP), Fmo2 (1:200; Proteintech, 67019- 1- Ig), Lcn2 (1:200; Proteintech, 26991- 1- AP), CD74 (1:200; Abcam, ab289885), troponin (1:200; Abcam, ab209813), CD31 (1:200; Abcam, ab182981), PDGFR (1:200; Abcam, ab203491), CD68 (1:200; Abcam, ab283654), and actin (1:200; Abcam, ab8227)]. .. The following day, horseradish peroxidase polymer–labeled secondary antibodies (1:200; Abcam, ab6721) were applied, followed by tyramide signal amplification (TSA) with TGseries tyramide fluorophores (TG 520N, TG 570N, TG 620N, TG 760N, and TG 700N) for signal amplification.
Article Title: Cytoskeletal remodeling promotes tunneling nanotube formation and drives cardiac resident cell mitochondrial transfer in sepsis
Article Snippet: Paraffin-embedded heart sections (5 μm) were deparaffinized, rehydrated through an ethanol gradient, and subjected to antigen retrieval using sodium citrate buffer (Servicebio, China). .. After blocking with goat serum, sections were incubated overnight at 4°C with primary antibodies [Cldn5 (1:200; Cell Signaling, #66879), Metal (:200; Abcam, ab12228), Cxcl2 (1:200; Abcam, ab317569), Gsn (1:200; Proteintech, 11644-2-AP), Fmo2 (1:200; Proteintech, 67019-1-Ig), Lcn2 (1:200; Proteintech, 26991-1-AP), CD74 (1:200; Abcam, ab289885), troponin (1:200; Abcam, ab209813), CD31 (1:200; Abcam, ab182981), PDGFR (1:200; Abcam, ab203491), CD68 (1:200; Abcam, ab283654), and actin (1:200; Abcam, ab8227)]. .. The following day, horseradish peroxidase polymer–labeled secondary antibodies (1:200; Abcam, ab6721) were applied, followed by tyramide signal amplification (TSA) with TG-series tyramide fluorophores (TG 520N, TG 570N, TG 620N, TG 760N, and TG 700N) for signal amplification.
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