among individual cells as an important regulatory step controlling
cellular processes [6]. Even subtle changes in RNA location and
copy number at a particular time are critical for deciding the fate of
a single cell [7]. To address this need, many in situ technologies
have been developed that allow imaging of mRNAs at a singlemolecule level, termed as single-molecule fluorescence in situ
hybridization (smFISH) [8]. smFISH detects single mRNAs
using an array of fluorescently labeled short oligonucleotide
(17–22 nt) probes that hybridize to the target [9]. This method
has been demonstrated to detect mRNA with high specificity in
bacteria, yeast [8], mammalian cells [10], C. elegans [8], neuron
cells [9], and single plant cells [11].
In this chapter, we describe an smFISH protocol that could be
used in highly autofluorescent plant tissues. This protocol introduces PHEM (PIPES, HEPES, EGTA, MgSO 4 ) buffer to the
fixation step to ensure the integrity of the treated plant cells. We
also introduce linear spectral unmixing and an emission fingerprinting imaging mode to eliminate the interference by plant autofluorescence [12]. Finally, the protocol includes an approach for
quantification of the smFISH signal in each sample.
2 Materials
Prepare all the solutions using Nanopure water. Do not use DEPCtreated water. All solutions should be stored at room temperature
(25
C) unless specified. Do not add Tween or other detergents to
the solutions.
2.1 Sample Fixation,
Embedding,
and Sectioning
1. PHEM buffer (2Â): 60 mM PIPES, 5 mM HEPES, 10 mM
EGTA, 2 mM MgSO 4 , pH 8. Weigh 18.14 g of PIPES, 6.5 g of
HEPES, 3.8 g EGTA, and 0.99 g MgSO 4 and add water to a
volume of 500 mL. Store at 4
C.
2. Fixation buffer: 4% Paraformaldehyde in PHEM buffer (1Â).
Add 1 ampoule (10 mL) of 16% paraformaldehyde to 30 mL
PHEM buffer (1Â). Fixation buffer should be prepared in a
chemical fume hood.
3. Water bath at 50
C.
4. Dissecting microscope.
5. Slide warmer.
6. Razor blades.
7. 20 mL Glass scintillation vials.
8. PBS buffer (10Â): Dissolve 80 g of NaCl, 2 g of KCl, 14.4 g of
Na 2 HPO 4 , and 2.4 g of KH 2 PO 4 . Adjust the pH to 7.1 and
bring volume to 1 L with water. Autoclave sterilize and store at
room temperature.
24
Kun Huang et al.
cellular processes [6]. Even subtle changes in RNA location and
copy number at a particular time are critical for deciding the fate of
a single cell [7]. To address this need, many in situ technologies
have been developed that allow imaging of mRNAs at a singlemolecule level, termed as single-molecule fluorescence in situ
hybridization (smFISH) [8]. smFISH detects single mRNAs
using an array of fluorescently labeled short oligonucleotide
(17–22 nt) probes that hybridize to the target [9]. This method
has been demonstrated to detect mRNA with high specificity in
bacteria, yeast [8], mammalian cells [10], C. elegans [8], neuron
cells [9], and single plant cells [11].
In this chapter, we describe an smFISH protocol that could be
used in highly autofluorescent plant tissues. This protocol introduces PHEM (PIPES, HEPES, EGTA, MgSO 4 ) buffer to the
fixation step to ensure the integrity of the treated plant cells. We
also introduce linear spectral unmixing and an emission fingerprinting imaging mode to eliminate the interference by plant autofluorescence [12]. Finally, the protocol includes an approach for
quantification of the smFISH signal in each sample.
2 Materials
Prepare all the solutions using Nanopure water. Do not use DEPCtreated water. All solutions should be stored at room temperature
(25
C) unless specified. Do not add Tween or other detergents to
the solutions.
2.1 Sample Fixation,
Embedding,
and Sectioning
1. PHEM buffer (2Â): 60 mM PIPES, 5 mM HEPES, 10 mM
EGTA, 2 mM MgSO 4 , pH 8. Weigh 18.14 g of PIPES, 6.5 g of
HEPES, 3.8 g EGTA, and 0.99 g MgSO 4 and add water to a
volume of 500 mL. Store at 4
C.
2. Fixation buffer: 4% Paraformaldehyde in PHEM buffer (1Â).
Add 1 ampoule (10 mL) of 16% paraformaldehyde to 30 mL
PHEM buffer (1Â). Fixation buffer should be prepared in a
chemical fume hood.
3. Water bath at 50
C.
4. Dissecting microscope.
5. Slide warmer.
6. Razor blades.
7. 20 mL Glass scintillation vials.
8. PBS buffer (10Â): Dissolve 80 g of NaCl, 2 g of KCl, 14.4 g of
Na 2 HPO 4 , and 2.4 g of KH 2 PO 4 . Adjust the pH to 7.1 and
bring volume to 1 L with water. Autoclave sterilize and store at
room temperature.
24
Kun Huang et al.