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 Chlorox 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 10X objective)

Fixative (25 mL)
Note: this gives you a final glutaraldehyde concentration of 1.25% Note: make this the same day
 * 20 mL of 200 mM cacodylate (pH 7.2)
 * 625 μL of 50% glutaraldehyde (in freezer)

X-gal solution (~5.6 mL)
Note: make this the same day
 * 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)

200 mM cacodylate, pH 7.2 (80 mL)
Note: this can be made in advance and stored in the refrigerator
 * 80 mL ddH2O
 * 2.56 g sodium cacodylate (Na+(CH3)2AsO2–)
 * pH to 7.2 with NaOH

100 mM potassium ferricyanide (20 mL)
Note: this can be made in advance and stored in the refrigerator
 * 20 mL ddH2O
 * 658.52 mg potassium ferricyanide (K3Fe(CN)6)

100 mM potassium ferrocyanide (20 mL)
Note: this can be made in advance and stored in the refrigerator
 * 20 mL ddH2O
 * 844.82 mg potassium hexacyanoferrate (II) trihydrate (K4Fe(CN)6·3H2O)

‡ 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.