All immunocytochemistry procedures were conducted at room temperature. Samples were first fixed with 4% PFA/PBS (v/v) solution for 15 mins and then permeabilized with 0.05% Triton-X/PBS (v/v) solution for 10 mins. Subsequently, samples were incubated for 1 hr with a monoclonal sarcomeric α-actinin (clone EA-53; Sigma-Aldrich) primary antibody, washed three times in PBS, and finally counterstained with: Alexa Fluor 488-conjugated anti-mouse secondary antibody, Alexa Fluor 633-conjugated Phalloidin and DAPI (Invitrogen). Samples were imaged using confocal microscopy, acquiring projected z-stack images of the wavy laminar tissues. The alignment and overall spatial organization of α-actinin positive structures in the immunostained digital images were evaluated with custom MATLAB (MathWorks, Natick, MA) code as previously described19 (link).
Alexa fluor 633 conjugated phalloidin and dapi
Manufactured by Thermo Fisher Scientific
Alexa Fluor 633-conjugated Phalloidin and DAPI are laboratory reagents used for fluorescence microscopy. Alexa Fluor 633-conjugated Phalloidin is a fluorescent stain that binds to F-actin, while DAPI is a fluorescent stain that binds to DNA. These reagents are commonly used together to visualize the cytoskeleton and nuclei of cells.
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2 protocols using alexa fluor 633 conjugated phalloidin and dapi
1
Quantitative Immunocytochemistry of Sarcomeric Structures
2
Quantitative Immunocytochemistry of Sarcomeric Structures
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All immunocytochemistry procedures were conducted at room temperature. Samples were first fixed with 4% PFA/PBS (v/v) solution for 15 mins and then permeabilized with 0.05% Triton-X/PBS (v/v) solution for 10 mins. Subsequently, samples were incubated for 1 hr with a monoclonal sarcomeric α-actinin (clone EA-53; Sigma-Aldrich) primary antibody, washed three times in PBS, and finally counterstained with: Alexa Fluor 488-conjugated anti-mouse secondary antibody, Alexa Fluor 633-conjugated Phalloidin and DAPI (Invitrogen). Samples were imaged using confocal microscopy, acquiring projected z-stack images of the wavy laminar tissues. The alignment and overall spatial organization of α-actinin positive structures in the immunostained digital images were evaluated with custom MATLAB (MathWorks, Natick, MA) code as previously described19 (link).
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