Arabidopsis thaliana Root Hair Cell Cytoplasmic pH (pHc) Imaging

Materials and Reagents

1. Stable Pt-GFP transgenic WT line N9561 (background: Col-0) was obtained from the Nottingham Arabidopsis Stock Centre http://arabidopsis.info/StockInfo?NASC_id=9561).
   Note: Pt-GFP T-DNA insertion mutant homozygous were obtained by crossing the T-DNA insertion mutant (what you interested, here meant the mutant with root hair growth problem) with the N9561. And we obtained the T-DNA insertion homozygous first by PCR (methods in http://signal.salk.edu/cgi-bin/tdnaexpress) in the F2 progeny, and we checked the fluorescence for Pt-GFP. Homozygous lines with fluorescence will be used for later analysis.
   
2. Digitonin (Sigma-Aldrich, catalog number: 11024-24-1 )
   Note: Risk assessment: toxic. Digitonin is toxic by inhalation, contact with skin, wear protective gloves and clothing, wear respiratory protection, when handling this chemical.
   
3. 2-(N-Morpholino) ethanesulfonic acid (MES) (Sigma-Aldrich, catalog number: 145224-94-8 )
   
4. N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: 7365-45-9 )
   
5. Murashige and Skoog (MS) medium (Phyto Tech, catalog number: M519 )
   
6. pH buffer solutions (see Recipes)
   

Equipment

1. Inverted Carl Zeiss LSM 710 confocal microscope
   
2. Plant growth chamber
   
3. 2 ml tubes (Eppendorf tubes)
   
4. Forceps
   
5. Microscope slides
   
6. Microscope cover slips
   

Software

1. Microsoft Excel

Procedure