Griffitts:Nodule Staining

Introduction

This method^‡ gives high-resolution view of infection thread networks (in young nodules) with no sectioning. Uptake of bacteria at the core of the young nodule is not observed in detail, but is inferred by hazy blue staining in that region. Mature nodules will have solid dark blue core. In infection mutants, staining is observed at the surface of the pseudonodule, but interior nodule tissue does not stain blue. The bleaching step greatly reduces opacity of the tissue. Based on my perusal of GUS protocols, this should be compatible, except that X-gal is simply replaced by X-gluc in the staining solution. However, most GUS protocols use phosphate buffer, maybe for historical reasons.

Procecure

• Inoculate germinated alfalfa in SNM tubes with a strain containing the hemA::lacZ construct pXLGD4
• Allow seven days for nodulation to occur before proceeding

Dissection

Note: This should be performed 7 days post-inoculation

• Prepare your fixative and X-gal solutions
• Fill labeled 60 mm Petri plates with 5 mL of dH₂O each
• Excise nodule-containing segments (1–2 cm) and store in Petri plates until all segments are dissected
• Remove plant from tube to a puddle of dH₂O on a glass plate
• Slice segments with a sterile razor blade while in contact with dH₂O

Fixation

• Replace water with 5 mL of fixative
• Pull vacuum for 15 minutes
  • Take care to avoid over-bubbling
  • Agitate roots to get them submerged
• Release vacuum
• Allow fixation to occur for an additional 60 minutes
• Rinse segments twice with 200 mM cacodylate (pH 7.2)
• Replace buffer with 2.5 mL of X-gal solution
• Parafilm
• Incubate at 30°C overnight
• Rinse segments twice with dH₂O
• Add 2 mL full-strength Clorox bleach
• Swirl segments for 45 seconds
• Immediately rinse twice with dH₂O
• Add 2 mL dH₂O
• Store segments in dH₂O at 4°C

Microscopy

Whole segments can be mounted in water (use silicone grease as padding) for viewing on the microscope (usually 10× objective)

Solutions

Fixative (25 mL)

• 20 mL of 200 mM cacodylate (pH 7.2)
• 625 μL of 50% glutaraldehyde (in freezer)

NOTE: this gives you a final glutaraldehyde concentration of 1.25%
NOTE: make this the same day

X-gal solution (~5.6 mL)

• 4.8 mL 200 mM cacodylate (pH 7.2)
• 300 μL 100 mM potassium ferricyanide
• 300 μL 100 mM potassium ferrocyanide
• 240 μL 2% X-gal

NOTE: make this the same day

200 mM cacodylate, pH 7.2 (80 mL)

• 80 mL ddH₂O
• 2.56 g sodium cacodylate (Na⁺(CH₃)₂AsO₂^–)
• pH to 7.2 with NaOH

NOTE: this can be made in advance and stored in the refrigerator

100 mM potassium ferricyanide (20 mL)

• 20 mL ddH₂O
• 658.52 mg potassium ferricyanide (K₃Fe(CN)₆)

NOTE: this can be made in advance and stored in the refrigerator

100 mM potassium ferrocyanide (20 mL)

• 20 mL ddH₂O
• 844.82 mg potassium hexacyanoferrate (II) trihydrate (K₄Fe(CN)₆·3H₂O)

NOTE: this can be made in advance and stored in the refrigerator

^‡ Compiled by Joel Griffitts; adapted from Boivin et al. (1990) "Rhizobium meliloti Genes Encoding Catabolism of Trigonelline are Induced under Symbiotic Conditions." Plant Cell, 2:1157–1170 PMID 12354952.