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Abstract Plant autophagy is a catabolic process where cellular components such as protein aggregates and dysfunctional organelles are degraded and recycled to maintain homeostasis and facilitate stress resilience. Autophagy relies on a double‐membrane vesicle called the autophagosome, which delivers cellular cargo to the vacuole for degradation. The Arabidopsis GFP‐ATG8 reporter line is a valuable tool widely used to visualize and quantify autophagosomes via microscopy and monitor autophagic degradation via immunoblotting. Consistent assessment of autophagic activity requires standardized protocols for sample preparation, imaging, and data analysis. Here, we present protocols for monitoring autophagy in Arabidopsis seedlings expressing GFP‐ATG8, including treatments to induce or inhibit autophagic flux, as well as imaging and image analysis procedures. These methods enable reliable evaluation of autophagic activity and can be adapted for diverse experimental conditions and genotypes. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Growth of Arabidopsis seedlings Basic Protocol 2: Activation of autophagy in Arabidopsis seedlings by abiotic stresses Basic Protocol 3: Inhibition of vacuolar degradation by concanamycin A treatment Basic Protocol 4: Quantification of GFP‐ATG8‐labeled autophagosomes in Arabidopsis seedlings via microscopy Basic Protocol 5: Analysis of autophagic degradation of GFP‐ATG8 via immunoblotting
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Insights into the Mechanical Characterization of Mouse Brain Tissue Using Microindentation Testing
Abstract Indentation testing is the most common approach to quantify mechanical brain tissue properties. Despite a myriad of studies conducted already, reported stiffness values vary extensively and continue to be subject of study. Moreover, the growing interest in the relationship between the brain's spatially heterogeneous microstructure and local tissue stiffness warrants the development of standardized measurement protocols to enable comparability between studies and assess repeatability of reported data. Here, we present three individual protocols that outline (1) sample preparation of a 1000‐µm thick coronal slice, (2) a comprehensive list of experimental parameters associated with the FemtoTools FT‐MTA03 Micromechanical Testing System for spherical indentation, and (3) two different approaches to derive the elastic modulus from raw force‐displacement data. Lastly, we demonstrate that our protocols deliver a robust experimental framework that enables us to determine the spatially heterogeneous microstructural properties of (mouse) brain tissue. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Mouse brain sample preparation Basic Protocol 2: Indentation testing of mouse brain tissue using the FemtoTools FT‐MTA03 Micromechanical Testing and Assembly System Basic Protocol 3: Tissue stiffness identification from force‐displacement data
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- Award ID(s):
- 2227232
- PAR ID:
- 10518373
- Publisher / Repository:
- Current Protocols
- Date Published:
- Journal Name:
- Current Protocols
- Volume:
- 4
- Issue:
- 4
- ISSN:
- 2691-1299
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1002/cpz1.1011
