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Leaf Clearing Protocol to Observe Stomata and Other Cells on Leaf Surface
Published: Sep 5, 2017 DOI: 10.21769/BioProtoc.2538 Views: 17541
Edited by: Scott A M McAdam
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Abstract
In this protocol, leaves are cleared and fixed in an ethanol and acetic acid solution, and mounted in Hoyer’s solution. The cleared leaves are imaged under differential interference contrast (DIC) microscope. This protocol is beneficial for studying stomata, hair cells, and other epidermal cells in plants.
Keywords: Leaf clearing
Stomata
DIC microscopy
Hoyer’s solution
Background
There are multiple ways to observe stomata and other epidermal cells such as hair cells on plant leaf surface. Traditionally a clear nail polish or wood glue is applied to the leaf surface and let dry. The leaf is peeled and observed under the microscope. Alternatively, scotch tape is applied to the leaf and removed to observe an imprint of the leaf surface. These traditional methods can be used for thicker leaves that are sturdy but the images are generally not of the highest quality. Small and delicate leaves such as Arabidopsis leaves require a more advanced method. A fresh Arabidopsis or Brachypodium leaf may also be observed directly under the microscope; however, the thickness and pigments in the leaf pose difficulties in viewing the stomata and other epidermal cells clearly. This protocol describes a method clearing of leaves for visualizing stomata including other epidermal cells and obtaining good quality images for publishing in peer-reviewed journals (Anderson, 1954).
Materials and Reagents
- Multi-well plates (Greiner Bio One International, catalog number: 665102 )
- Parafilm (Bemis, catalog number: PM996 )
- Microscope slides, 25 x 75 x 1.0 mm (Fisher Scientific, catalog number: 12-550-A3 )
- Cover slips (Fisher Scientific, catalog number: 12-544D )
- Aluminium foil
- Arabidopsis or Brachypodium leaf
Note: You may cut the Brachypodium leaf into 1 cm pieces to fit into wells. - Ethanol (Sigma-Aldrich, catalog number: 459844 )
- Acetic acid (Sigma-Aldrich, catalog number: AX0077-1 )
- Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 221473 )
- Gum Arabic (Sigma-Aldrich, catalog number: 30888-1KG )
- Chloral hydrate (Sigma-Aldrich, catalog number: C8383 )
- Glycerol (Sigma-Aldrich, catalog number: G5516-1L )
Note: Sterile is not required. - Distilled water
- Clearing solution (see Recipes)
- 1 N KOH (see Recipes)
- Hoyer’s solution (see Recipes)
Equipment
- Fume hood
- Forceps (Fisher Scientific, catalog number: 22-327379 )
- Microscope (Leica, model: Leica DM6 B )
- 1 L glass beaker (Sigma-Aldrich, catalog number: Z169161 )
- Magnetic stirrer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: S194615 )
Software
- MS Excel
(https://support.office.com/en-us/article/STDEV-function-51fecaaa-231e-4bbb-9230-33650a72c9b0)
Note: Calculate Standard Deviation in MS Excel.
Procedure
Category
Plant Science > Plant cell biology > Cell isolation
Cell Biology > Cell imaging > Fixed-tissue imaging
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