Autophagosome and Autolysosome Identification

Since RFP (pKa 4.5) fluorescence is stable in an acidic environment, but GFP (pKa5.9) fluorescence is quenched in the acidic lysosomal compartment [26 (link)], autophagosomes (yellow) can be distinguished from autolysosomes (red), and flux can be determined directly. RFP/GFP analysis was performed as described [19 (link)]. Briefly, hearts were embedded in freezing matrix (OCT), and 8 μm frozen sections were prepared on a Leica CM3000 Cryostat (Leica Microsystems, Buffalo Grove, IL). Images were acquired on a fluorescent microscope.

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Zheng D., Zhang Y., Zheng M., Cao T., Wang G., Zhang L., Ni R., Brockman J., Zhong H., Fan G.C, & Peng T. (2019). Nicotinamide riboside promotes autolysosome clearance in preventing doxorubicin-induced cardiotoxicity. Clinical science (London, England : 1979), 133(13), 1505-1521.

Publication 2019

Leica CM3000 Cryostat

Acidic Autolysosomes Autophagosomes Buffalo Fluorescence Frozen sections Hearts Lysosomal Microscope

Corresponding Organization : Western University

Other organizations : Canada Research Chairs, University of Toronto, University of Cincinnati Medical Center

Top 5 similar protocols

Variable analysis

independent variables

Presence of RFP (pKa 4.5) and GFP (pKa 5.9) fluorescence

dependent variables

Distinction between autophagosomes (yellow) and autolysosomes (red)
Autophagy flux

control variables

Frozen sections prepared on a Leica CM3000 Cryostat
Images acquired on a fluorescent microscope

controls

No positive or negative controls were explicitly mentioned in the provided information.

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!