PEA-CLARITY: Three Dimensional (3D) Molecular Imaging of Whole Plant Organs
全植株的PEA-CLARITY三维分子成像
发布: 2016年11月05日第6卷第21期 DOI: 10.21769/BioProtoc.2000 浏览次数: 9867
评审: Arsalan DaudiTeresa LenserAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Sep 2015
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Abstract
Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration. Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in tobacco and Arabidopsis leaves. Enzyme treatment enabled penetration of antibodies into whole tissues without the need for any sectioning of the material. Therefore, this protocol facilitates protein localisation of intact tissue in 3D whilst retaining cellular structure.
Background
Fixation and embedding of plant tissue for molecular interrogation using techniques such as histological staining, immunohistochemistry or in situ hybridisation has been the foundation of cell biology studies for decades. Applying these techniques for 3D tissue analysis is seriously limited by the need to section the tissue, image each section, and then reassemble the images into a 3D representation of the structures of interest. Here we present a fundamental shift from the two dimensional plane to that of three dimensions whilst retaining molecular structures of interest without the need to section the plant tissue. Recent advances in fixation and ‘clearing’ techniques such as SeeDB, ScaleA2, 3DISCO, CLARITY and its recent variant PACT enabled intact imaging of whole embryos, brains and other organs in mouse and rat models. The new CLARITY system fixes and binds tissues within an acrylamide mesh structure. Proteins and nucleic acids are covalently linked to the acrylamide mesh by formaldehyde, then optically interfering lipid structures of animal cell membranes are removed using detergent (SDS). This renders such tissue optically transparent and suitable for deep imaging of up to ~5 mm using confocal microscopy.
Materials and Reagents
- 50 ml conical tube
- 1.5 ml microfuge tubes
- Aluminum foil
- Parafilm (Sigma-Aldrich, catalog number: P-7793 )
- Lint free paper
- 1.5 ml Protein LoBind tubes (Eppendorf, catalog number: 0030108116 )
- Glass microscope slide
- Glass microscope coverslip
- Nicotiana tabacum (Hanson and Köhler, 2001)
- 16% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15710 )
- Sodium azide (NaN3) (Sigma-Aldrich, catalog number: S-2002 )
- 0.005% NaN3 in PBS (N3PBS)
- Triton X-100 (Sigma-Aldrich, catalog number: T-9284 )
- Dulbecco's phosphate buffered saline (DPBS, autoclaved) (Thermo Fisher Scientific, GibcoTM, 21600-010 )
- 0.1% Triton X-100 in PBS (PBST)
- Vaseline (Unilever, VASELINE®)
- BluTack (Bostic)
- Rubisco antibody (rabbit) (Gift - Spencer Whitney, Whitney and Andrews, 2001)
- Cy5 secondary AB (anti-rab) (Abcam, catalog number: Ab6564 )
- Propidium iodide (Sigma-Aldrich, catalog number: P-4864 )
- Calcofluor white (Sigma-Aldrich, catalog number: F-3543 )
- 40% acrylamide (Bio-Rad Laboratories, catalog number: 161-0140 )
- 2% bis acrylamide (Bio-Rad Laboratories, catalog number: 161-0142 )
- VA-044 initiator (Wako Pure Chemical Industries, catalog number: 017-19362 )
- Deionized and distilled water (ddH2O)
- Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L-3771 )
- Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B-6768 )
- Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S-8045 )
- α-amylase (Megazyme, catalog number: E-ANAAM )
- α-L-arabinofuranosidase (Megazyme, catalog number: E-ABFCJ )
- β-mannanase (Megazyme, catalog number: E-BMACJ )
- Cellulase (Megazyme, catalog number: E-CELBA )
- Pectate lyase (Megazyme, catalog number: E-PLYCJ )
- Xyloglucanase (Megazyme, catalog number: E-XEGP )
- Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C-5670 )
- Hydrogel solution (200 ml) (see Recipes)
- SDS clearing solution (1 L) (see Recipes)
- Enzyme treatment solution (10 ml) (see Recipes)
Notes:
- Please refer to MSDS before conducting protocol as paraformaldehyde (PFA), acrylamide, sodium dodecyl sulfate (SDS) and sodium azide (NaN3) are known irritants, sensitizers, carcinogens and neurotoxins. The use of personal protective equipment (PPE) is imperative whilst undertaking this protocol.
- Any specific IgG primary antibody and respective secondary antibody can be used with this protocol.
- Protocol can be paused and samples stored at any stage from step D onwards in either SDS clearing solution or N3PBS.
Equipment
- Vacuum pump at -100 kPa
- Fume hood
- 4 °C fridge
- 37 °C water bath
- Weigh balance
- 37 °C incubator shaker
- Leica SP8 confocal microscope/lightsheet microscope or equivalent
- Long working distance objectives greater than 2 mm
Software
- Leica Applications Suite - Fluorescence (LAS-AF) software
Procedure
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文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Palmer, W. M., Martin, A. P., Flynn, J. R., Reed, S., White, R., Furbank, R. T. and Grof, C. P. L. (2016). PEA-CLARITY: Three Dimensional (3D) Molecular Imaging of Whole Plant Organs. Bio-protocol 6(21): e2000. DOI: 10.21769/BioProtoc.2000.
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分类
植物科学 > 植物细胞生物学 > 细胞成像
植物科学 > 植物生理学 > 表型分析
细胞生物学 > 细胞成像 > 活细胞成像
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