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Title: Staining and automated image quantification of callose in Arabidopsis cotyledons and leaves.
Callose is a β-1,3-glucan polysaccharide that is deposited at discrete sites in the plant cell wall in response to microbial pathogens, likely contributing to protection against pathogen infection. Increased callose deposition also occurs in response to the 22-amino acid peptide flg22, a pathogen-associated molecular pattern (PAMP) derived from bacterial flagellin protein. Here, we provide protocols for callose staining using aniline blue in cotyledon and leaf tissue of the model plant Arabidopsis thaliana. Aniline blue stain utilizes a fluorochrome that complexes with callose for its visualization by microscopy using an ultraviolet (UV) filter. For robust quantification of callose deposits, we outline an automated image analysis workflow utilizing the freely available Fiji (Fiji Is Just ImageJ; NIH) software and a Trainable Weka Segmentation (TWS) plugin. Our methodology for automated analysis of large batches of images can be easily adapted to quantify callose in other tissues and plant species, as well as to quantify fluorescent structures other than callose.  more » « less
Award ID(s):
1758843
PAR ID:
10296975
Author(s) / Creator(s):
; ;
Editor(s):
Anderson, CT; Elizabeth S. Haswell, ES; null
Date Published:
Journal Name:
Methods in cell biology
Volume:
160
ISSN:
0091-679X
Page Range / eLocation ID:
181 - 199
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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