similar approach may be taken for analysis in other tissues. Importantly, the methods described here are not specific to the lacO/LacI
system but could be readily adapted to other locus-tagging
approaches.
2 Materials
2.1 Plant Growth
1. Arabidopsis lines with a locus-tagging system (lacO/LacI system see Note 1).
2. Murashige & Skoog basal medium with vitamins (later referred
as MS medium).
3. 5% Bleach (hypochlorite) in dH 2 O.
4. Plant growth chamber (see Note 2).
5. Square Petri dishes (see Note 3).
6. Adhesive tape.
7. Laminar flow cabinet.
8. Sterile razor blades.
9. Coverslips No 1.5.
10. Microscope slides with frosted ends.
11. Secure Seal Adhesive Sheets (0.12 mm thick) (see Note 4).
2.2 Microscopy
1. Laser scanning confocal microscope (see Note 5).
2. Objective 63Â/1.2 water or another high numerical aperture
water immersion objective (see Note 6).
3. An argon 488 nm laser line is required for GFP.
2.3 Data Analysis
1. ImageJ or Fiji software (NIH, Bethesda, MD, http://rsb.info.
nih.gov/ij/) with the plugin SpotTracker 2D (obtained from
http://bigwww.epfl.ch/sage/soft/spottracker).
2. Software to perform data analysis (mean intensity values, data
plotting and curve fitting with best-fit equations): Microsoft
Excel, GraphPad Software (La Jolla California USA, www.gra
phpad.com), MATLAB, or alike.
3 Methods
3.1 Plant Growth
and Sample
Preparation
The objective of this part of the protocol is to grow seedlings with
easy access to root tissue, which will then be transferred to a
microscope slide for imaging.
1. Surface-sterilize seeds in 5% v/v sodium hypochlorite for 5 min
and rinse three times in sterile distilled water.
Visualizing and Measuring Single Locus Dynamics in Arabidopsis thaliana
215
system but could be readily adapted to other locus-tagging
approaches.
2 Materials
2.1 Plant Growth
1. Arabidopsis lines with a locus-tagging system (lacO/LacI system see Note 1).
2. Murashige & Skoog basal medium with vitamins (later referred
as MS medium).
3. 5% Bleach (hypochlorite) in dH 2 O.
4. Plant growth chamber (see Note 2).
5. Square Petri dishes (see Note 3).
6. Adhesive tape.
7. Laminar flow cabinet.
8. Sterile razor blades.
9. Coverslips No 1.5.
10. Microscope slides with frosted ends.
11. Secure Seal Adhesive Sheets (0.12 mm thick) (see Note 4).
2.2 Microscopy
1. Laser scanning confocal microscope (see Note 5).
2. Objective 63Â/1.2 water or another high numerical aperture
water immersion objective (see Note 6).
3. An argon 488 nm laser line is required for GFP.
2.3 Data Analysis
1. ImageJ or Fiji software (NIH, Bethesda, MD, http://rsb.info.
nih.gov/ij/) with the plugin SpotTracker 2D (obtained from
http://bigwww.epfl.ch/sage/soft/spottracker).
2. Software to perform data analysis (mean intensity values, data
plotting and curve fitting with best-fit equations): Microsoft
Excel, GraphPad Software (La Jolla California USA, www.gra
phpad.com), MATLAB, or alike.
3 Methods
3.1 Plant Growth
and Sample
Preparation
The objective of this part of the protocol is to grow seedlings with
easy access to root tissue, which will then be transferred to a
microscope slide for imaging.
1. Surface-sterilize seeds in 5% v/v sodium hypochlorite for 5 min
and rinse three times in sterile distilled water.
Visualizing and Measuring Single Locus Dynamics in Arabidopsis thaliana
215