Blocking Assay:Article Title: Immunoglobulin A/PIGR axis as potential mediators of human abdominal aortic aneurysms revealed by topologically resolved proteomics.
Article Snippet: After determining protein concentration using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), equal amounts of protein (20 μg) were subjected to sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and subsequently transferred to a nitrocellulose membrane (Bio-Rad). .. After blocking with 5% BSA in TBS containing 0.1% Tween-20, blots were incubated overnight with one of the following antibodies: anti-human IGA1 and IGA2 (9130-01 and 9140-01, Southern biotech), PIGR (AF2717, R&D) or anti-human GAPDH (MAB374, Sigma) at 4 °C. .. Blots were then washed and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for one hour and visualized using the ECL substrate kit (Thermo Fisher Scientific).
Article Title: Immunoglobulin A/PIGR axis as potential mediators of human abdominal aortic aneurysms revealed by topologically resolved proteomics
Article Snippet: After determining protein concentration using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), equal amounts of protein (20 μg) were subjected to sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and subsequently transferred to a nitrocellulose membrane (Bio-Rad). .. After blocking with 5% BSA in TBS containing 0.1% Tween-20, blots were incubated overnight with one of the following antibodies: anti-human IGA1 and IGA2 (9130-01 and 9140-01, Southern biotech), PIGR (AF2717, R&D) or anti-human GAPDH (MAB374, Sigma) at 4 °C. .. Blots were then washed and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for one hour and visualized using the ECL substrate kit (Thermo Fisher Scientific).
Incubation:Article Title: Immunoglobulin A/PIGR axis as potential mediators of human abdominal aortic aneurysms revealed by topologically resolved proteomics.
Article Snippet: After determining protein concentration using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), equal amounts of protein (20 μg) were subjected to sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and subsequently transferred to a nitrocellulose membrane (Bio-Rad). .. After blocking with 5% BSA in TBS containing 0.1% Tween-20, blots were incubated overnight with one of the following antibodies: anti-human IGA1 and IGA2 (9130-01 and 9140-01, Southern biotech), PIGR (AF2717, R&D) or anti-human GAPDH (MAB374, Sigma) at 4 °C. .. Blots were then washed and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for one hour and visualized using the ECL substrate kit (Thermo Fisher Scientific).
Article Title: Immunoglobulin A/PIGR axis as potential mediators of human abdominal aortic aneurysms revealed by topologically resolved proteomics
Article Snippet: After determining protein concentration using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), equal amounts of protein (20 μg) were subjected to sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and subsequently transferred to a nitrocellulose membrane (Bio-Rad). .. After blocking with 5% BSA in TBS containing 0.1% Tween-20, blots were incubated overnight with one of the following antibodies: anti-human IGA1 and IGA2 (9130-01 and 9140-01, Southern biotech), PIGR (AF2717, R&D) or anti-human GAPDH (MAB374, Sigma) at 4 °C. .. Blots were then washed and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for one hour and visualized using the ECL substrate kit (Thermo Fisher Scientific).
Article Title: Viral vector delivered immunogen focuses HIV-1 antibody specificity and increases durability of the circulating antibody recall response
Article Snippet: Briefly, plasma samples were diluted at 1:50 (for IgG) or 1:40 (for IgG1-IgG4 and IgA1-IgA2 subclasses) and incubated with carboxylated fluorescent bead sets (Bio-Rad) covalently coupled to Env proteins. .. HIV-1-specific antibody subclasses were detected with biotinylated mouse anti-human IgG (Southern Biotech), IgG1 (BD Pharmingen), IgG2 (Southern Biotech), IgG3 (Calbiochem), IgG4 (BD Pharmingen), IgA1 (Southern Biotech), or IgA2 (Southern Biotech), followed by washing and incubation with streptavidin phycoerythrin (BD Pharmingen). .. Beads were acquired on a Bio-Plex 200 instrument (Bio-Rad, Inc.), with antibody binding expressed as mean fluorescence intensity (MFI).
Article Title: Immunoglobulin A/PIGR axis as potential mediators of human abdominal aortic aneurysms revealed by topologically resolved proteomics
Article Snippet: .. For immunohistochemical analysis of IGA1, IGA2 and PIGR and for immunofluorescence analysis of PIGR in aortic paraffin sections, antigen retrieval (0.01 M citrate pH 6, 96 °C, 20 min), blockade of endogenous peroxidase (3% H 2 O 2 in methanol, 30 min) and nonspecific binding (6% host serum, 1 h) was performed prior to incubation with the primary antibodies overnight at 4 °C (anti-human IGA1 and IGA2, 9130–01 and 9140–01, respectively, Southern Biotech, and anti-human PIGR, ab96196, Abcam)Then, samples were rinsed in PBS and incubated 1 h with goat anti-mouse and donkey anti-rabbit (HRP-conjugated, Jackson Immunoresearch) secondary antibodies. .. Slides were then incubated with 3,3'-diaminobenzidine (DAB) peroxidase substrate, counterstained with hematoxylin, dehydrated, mounted with DPX and examined under a light microscope (Zeiss).
Article Title: Immunoglobulin A/PIGR axis as potential mediators of human abdominal aortic aneurysms revealed by topologically resolved proteomics.
Article Snippet: .. For immunohistochemical analysis of IGA1, IGA2 and PIGR and for immunofluorescence analysis of PIGR in aortic paraffin sections, antigen retrieval (0.01 M citrate pH 6, 96 °C, 20 min), blockade of endogenous peroxidase (3% H2O2 in methanol, 30 min) and nonspecific binding (6% host serum, 1 h) was performed prior to incubation with the primary antibodies overnight at 4 °C (antihuman IGA1 and IGA2, 9130–01 and 9140–01, respectively, Southern Biotech, and anti-human PIGR, ab96196, Abcam)Then, samples were rinsed in PBS and incubated 1 h with goat anti-mouse and donkey anti-rabbit (HRP-conjugated, Jackson Immunoresearch) secondary antibodies. .. Slides were then incubated with 3,3’-diaminobenzidine (DAB) peroxidase substrate, counterstained with hematoxylin, dehydrated, mounted with DPX and examined under a light microscope (Zeiss).
Immunohistochemical staining:Article Title: Immunoglobulin A/PIGR axis as potential mediators of human abdominal aortic aneurysms revealed by topologically resolved proteomics
Article Snippet: .. For immunohistochemical analysis of IGA1, IGA2 and PIGR and for immunofluorescence analysis of PIGR in aortic paraffin sections, antigen retrieval (0.01 M citrate pH 6, 96 °C, 20 min), blockade of endogenous peroxidase (3% H 2 O 2 in methanol, 30 min) and nonspecific binding (6% host serum, 1 h) was performed prior to incubation with the primary antibodies overnight at 4 °C (anti-human IGA1 and IGA2, 9130–01 and 9140–01, respectively, Southern Biotech, and anti-human PIGR, ab96196, Abcam)Then, samples were rinsed in PBS and incubated 1 h with goat anti-mouse and donkey anti-rabbit (HRP-conjugated, Jackson Immunoresearch) secondary antibodies. .. Slides were then incubated with 3,3'-diaminobenzidine (DAB) peroxidase substrate, counterstained with hematoxylin, dehydrated, mounted with DPX and examined under a light microscope (Zeiss).
Article Title: Immunoglobulin A/PIGR axis as potential mediators of human abdominal aortic aneurysms revealed by topologically resolved proteomics.
Article Snippet: .. For immunohistochemical analysis of IGA1, IGA2 and PIGR and for immunofluorescence analysis of PIGR in aortic paraffin sections, antigen retrieval (0.01 M citrate pH 6, 96 °C, 20 min), blockade of endogenous peroxidase (3% H2O2 in methanol, 30 min) and nonspecific binding (6% host serum, 1 h) was performed prior to incubation with the primary antibodies overnight at 4 °C (antihuman IGA1 and IGA2, 9130–01 and 9140–01, respectively, Southern Biotech, and anti-human PIGR, ab96196, Abcam)Then, samples were rinsed in PBS and incubated 1 h with goat anti-mouse and donkey anti-rabbit (HRP-conjugated, Jackson Immunoresearch) secondary antibodies. .. Slides were then incubated with 3,3’-diaminobenzidine (DAB) peroxidase substrate, counterstained with hematoxylin, dehydrated, mounted with DPX and examined under a light microscope (Zeiss).
Immunofluorescence:Article Title: Immunoglobulin A/PIGR axis as potential mediators of human abdominal aortic aneurysms revealed by topologically resolved proteomics
Article Snippet: .. For immunohistochemical analysis of IGA1, IGA2 and PIGR and for immunofluorescence analysis of PIGR in aortic paraffin sections, antigen retrieval (0.01 M citrate pH 6, 96 °C, 20 min), blockade of endogenous peroxidase (3% H 2 O 2 in methanol, 30 min) and nonspecific binding (6% host serum, 1 h) was performed prior to incubation with the primary antibodies overnight at 4 °C (anti-human IGA1 and IGA2, 9130–01 and 9140–01, respectively, Southern Biotech, and anti-human PIGR, ab96196, Abcam)Then, samples were rinsed in PBS and incubated 1 h with goat anti-mouse and donkey anti-rabbit (HRP-conjugated, Jackson Immunoresearch) secondary antibodies. .. Slides were then incubated with 3,3'-diaminobenzidine (DAB) peroxidase substrate, counterstained with hematoxylin, dehydrated, mounted with DPX and examined under a light microscope (Zeiss).
Article Title: Immunoglobulin A/PIGR axis as potential mediators of human abdominal aortic aneurysms revealed by topologically resolved proteomics.
Article Snippet: .. For immunohistochemical analysis of IGA1, IGA2 and PIGR and for immunofluorescence analysis of PIGR in aortic paraffin sections, antigen retrieval (0.01 M citrate pH 6, 96 °C, 20 min), blockade of endogenous peroxidase (3% H2O2 in methanol, 30 min) and nonspecific binding (6% host serum, 1 h) was performed prior to incubation with the primary antibodies overnight at 4 °C (antihuman IGA1 and IGA2, 9130–01 and 9140–01, respectively, Southern Biotech, and anti-human PIGR, ab96196, Abcam)Then, samples were rinsed in PBS and incubated 1 h with goat anti-mouse and donkey anti-rabbit (HRP-conjugated, Jackson Immunoresearch) secondary antibodies. .. Slides were then incubated with 3,3’-diaminobenzidine (DAB) peroxidase substrate, counterstained with hematoxylin, dehydrated, mounted with DPX and examined under a light microscope (Zeiss).
Binding Assay:Article Title: Immunoglobulin A/PIGR axis as potential mediators of human abdominal aortic aneurysms revealed by topologically resolved proteomics
Article Snippet: .. For immunohistochemical analysis of IGA1, IGA2 and PIGR and for immunofluorescence analysis of PIGR in aortic paraffin sections, antigen retrieval (0.01 M citrate pH 6, 96 °C, 20 min), blockade of endogenous peroxidase (3% H 2 O 2 in methanol, 30 min) and nonspecific binding (6% host serum, 1 h) was performed prior to incubation with the primary antibodies overnight at 4 °C (anti-human IGA1 and IGA2, 9130–01 and 9140–01, respectively, Southern Biotech, and anti-human PIGR, ab96196, Abcam)Then, samples were rinsed in PBS and incubated 1 h with goat anti-mouse and donkey anti-rabbit (HRP-conjugated, Jackson Immunoresearch) secondary antibodies. .. Slides were then incubated with 3,3'-diaminobenzidine (DAB) peroxidase substrate, counterstained with hematoxylin, dehydrated, mounted with DPX and examined under a light microscope (Zeiss).
Article Title: Immunoglobulin A/PIGR axis as potential mediators of human abdominal aortic aneurysms revealed by topologically resolved proteomics.
Article Snippet: .. For immunohistochemical analysis of IGA1, IGA2 and PIGR and for immunofluorescence analysis of PIGR in aortic paraffin sections, antigen retrieval (0.01 M citrate pH 6, 96 °C, 20 min), blockade of endogenous peroxidase (3% H2O2 in methanol, 30 min) and nonspecific binding (6% host serum, 1 h) was performed prior to incubation with the primary antibodies overnight at 4 °C (antihuman IGA1 and IGA2, 9130–01 and 9140–01, respectively, Southern Biotech, and anti-human PIGR, ab96196, Abcam)Then, samples were rinsed in PBS and incubated 1 h with goat anti-mouse and donkey anti-rabbit (HRP-conjugated, Jackson Immunoresearch) secondary antibodies. .. Slides were then incubated with 3,3’-diaminobenzidine (DAB) peroxidase substrate, counterstained with hematoxylin, dehydrated, mounted with DPX and examined under a light microscope (Zeiss).
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