Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides
拟南芥粘液多糖的全种子免疫标记
发布: 2017年06月05日第7卷第11期 DOI: 10.21769/BioProtoc.2323 浏览次数: 8411
评审: Marisa RosaAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Dec 2015
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Abstract
In addition to synthesizing and secreting copious amounts of pectic polymers (Young et al., 2008), Arabidopsis thaliana seed coat epidermal cells produce small amounts of cellulose and hemicelluloses typical of secondary cell walls (Voiniciuc et al., 2015c). These components are intricately linked and are released as a large mucilage capsule upon hydration of mature seeds. Alterations in the structure of minor mucilage components can have dramatic effects on the architecture of this gelatinous cell wall. The immunolabeling protocol described here makes it possible to visualize the distribution of specific polysaccharides in the seed mucilage capsule.
Keywords: Arabidopsis thaliana (拟南芥)
Plant cell wall (植物细胞壁)
Immunolabeling (免疫标记)
Carbohydrate (糖类)
Fluorescence (荧光)
Seed coat (种皮)
Microscopy (显微镜检查)
Background
Since the first comprehensive immunofluorescence analysis of pectin-rich mucilage in Arabidopsis seed coat epidermal cells (Young et al., 2008), additional types of polysaccharides have been detected in this specialized cell wall (Voiniciuc et al., 2015a; 2015b and 2015c). To handle more samples in parallel, I adapted the original protocols (performed in 1.5-ml microcentrifuge tubes; Young et al., 2008; Harpaz-Saad et al., 2011) to a 24-well plate format. I recommend counterstaining seeds with Pontamine S4B, a fluorescent dye that is more specific to cellulose than previous stains (Anderson et al., 2010). By testing for cross-talk between multiple fluorophores and setting clear guidelines for image acquisition and processing, this protocol yields reproducible mucilage phenotypes that can be reliably interpreted.
Materials and Reagents
- Personal protection equipment (safety glasses, lab coat, gloves)
- 1.5 ml snap-cap tubes
- 24-well plate with lid (VWR, catalog number: 734-2325 )
- Plastic Pasteur pipette (VWR, catalog number: LSUK711117S/20 )
- Manual pipettes tips (Eppendorf, Research plus and Repeater plus, or similar style)
- Aluminum foil
- MARIENFELD glass slides, low autofluorescence, L x B x H: 76 x 26 x 1 mm; pre-washed; Ground edges, 90° slides (VWR, catalog number: 631-9464 )
- Precision cover slip glass, thickness No. 1.5H (Marienfeld-Superior, catalog number: 0107222 )
- 15 ml Falcon tubes (VWR, catalog number: 734-0452 )
- Arabidopsis thaliana mature, dry seeds
- Primary antibodies directed against plant cell wall carbohydrates, obtained from:
- CarboSource (http://www.ccrc.uga.edu/~carbosource/CSS_home.html)
- Plant probes (http://www.plantprobes.net/index.php)
- Alexa Fluor® 488 secondary antibodies (against the host of the primary antibody)
- Goat anti-mouse (Thermo Fisher Scientific, Invitrogen, catalog number: A-11001 )
- Goat anti-rat (Thermo Fisher Scientific, Invitrogen, catalog number: A-11006 )
- Clear nail polish
- Sodium phosphate, dibasic (Na2HPO4·2H2O) (Carl Roth, catalog number: 4984.2 )
- Sodium phosphate, monobasic (NaH2PO4·2H2O) (Carl Roth, catalog number: T879.2 )
- Bovine serum albumin (BSA), IgG free (Carl Roth, catalog number: 3737.2 )
- Pontamine S4B (sold as Direct Red 23) (Sigma-Aldrich, catalog number: 212490-50G )
- Sodium chloride (NaCl) (Carl Roth, catalog number: P029.1 )
- Phosphate-buffered saline (PBS), pH 7.0 (see Recipes)
- Blocking solution (see Recipes)
- Antibody solution (see Recipes)
- 0.01% (w/v) S4B in 50 mM NaCl (see Recipes)
Equipment
- Manual pipettes (Eppendorf, models: Research® plus and Repeater® plus ; or similar style)
- Water purification system (Milli-Q or similar style)
- Balance
- Autoclave
- Orbital shaker
- Vortex mixer (Scientific Industries, model: Vortex-Genie 2 ; or similar style)
- Leica TCS SP8 confocal microscope (Leica Microsystems, model: TCS SP8 ) with the following key components:
- DM 5000 upright microscope with manual stage
- HC PL APO 10x/0.40 CS (Leica Microsystems, catalog number: 15506285 ) objective
- Compact LIAchroic AOTF unit with 488 nm and 552 nm solid state lasers
- SP8 Scan Head with LIAchroic beam splitters for filter-free spectral detection
Note: Alternatively, use a conventional confocal microscope equipped with filter-based detection. Filters designed for green fluorescent proteins (GFP) and red fluorescent proteins (RFP) are generally suitable for the acquisition of Alexa Fluor® 488 and Pontamine S4B signals, respectively. In this protocol, the emission of fluorophores was detected in 500-530 nm and 590-700 nm ranges. - Two fluorescence photomultipliers and one transmitted light detector
- Scan optics module HIVIS with rotation
- Computer for image acquisition and processing
Software
- Leica Application Suite X (LAS X) software for image acquisition
- Fiji is just ImageJ (Fiji) image processing package (https://fiji.sc/)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Voiniciuc, C. (2017). Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides. Bio-protocol 7(11): e2323. DOI: 10.21769/BioProtoc.2323.
- Voiniciuc, C., Gunl, M., Schmidt, M. H. and Usadel, B. (2015). Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds. Plant Physiol 169(4): 2481-2495.
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分类
植物科学 > 植物生物化学 > 糖类
细胞生物学 > 组织分析 > 组织染色
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