Mouse hearts were sectioned on a coronal plane and frozen in optimal cutting temperature medium. Cryostat sections (≈5 μm thick) were affixed to glass slides, postfixed in fresh 4% paraformaldehyde, and permeabilized with 0.2% (v/v) Triton X‐100. Slides were blocked, washed, and incubated overnight at room temperature in a humidified chamber with antibodies diluted in blocking buffer (Table S1 ). After washing, sections were incubated for 90 minutes at room temperature with secondary antibodies diluted in blocking buffer. After washing, slides were mounted with a coverslip using Prolong Diamond (ThermoFisher, P36961) and imaged using a Leica confocal microscope (LSM510) with the ×60 oil objective.
Confocal microscope lsm510
Manufactured by Leica
The Leica confocal microscope (LSM510) is a high-performance imaging system designed for advanced microscopy applications. It features a confocal scanning mechanism that allows for optical sectioning and high-resolution imaging of samples. The LSM510 provides researchers with the ability to capture detailed, three-dimensional images of various specimens.
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2 protocols using confocal microscope lsm510
1
Immunofluorescence of Mouse Heart Sections
2
Immunohistochemical Analysis of Mouse Hearts
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Mouse hearts were sectioned on a coronal plane and frozen in OCT medium. Cryostat sections (~5 μm thick) were affixed to glass slides, postfixed in fresh 4% paraformaldehyde, and permeabilized with 0.2% (v/v) TritonX-100. Slides were blocked, washed, and incubated Page 10 of 49 overnight at room temperature in a humidified chamber with antibodies diluted in blocking buffer (Supp. Table 1). After washing, sections were incubated for 90 mins at room temperature with secondary antibodies diluted in blocking buffer. After washing, slides were mounted with a coverslip using Prolong Diamond (ThermoFisher, P36961) and imaged using a Leica confocal microscope (LSM510) with the 60x oil objective.
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