A. Preparation of tissue
  1. Obtain normal plant tissue. Cut material may be kept in water for up to an hour in many cases.
  2. Pieces of tissue should be dissected using a new double edge razor blade or similarly sharp object using a dissecting microscope. Tissue should be thinner than 1 mm in at least one dimension.
B. Fixation
  1. Transfer tissue to a vial of 3% glutaraldehyde in 0.1 M cacodylate at pH 7.4. Sucrose in the buffer is not usually necessary, but may be beneficial in preserving senescent tissue. (Since the initial pH of the plant vacuole is low, some workers use a higher pH to fix - up to 7.8). Fix 2 hours to overnight. Air in intercellular spaces may have to be removed during fixation using a partial vacuum to remove the air. After two hours, transfer material to refrigerator at 4° C.
  2. Tissue may be processed immediately or held up to several months in a holding solution of 0.1 M cacodylate - 0.1 M sucrose buffer at pH of fixation at 4° C.
  3. Rinse tissue with three changes of cold buffer, 15 minutes per change.
  4. Postfix in cold 2% osmium tetroxide (mixed 1:1 using 4% aqueous osmium and 2X buffer) for 2 hours on ice or in fridge. Work with uncapped osmium solutions only under the fume hood.
  5. Rinse tissue with cold buffer for 15 minutes.
C. Dehydration
  1. Dehydrate the tissue using the following concentrations of ethanol in a cold graded ethanol series (15 - 30 minutes per change): 30%, 50%, 70%, 80%, 90%, 100%. (Cold dehydration fluids tend to lend some rigidity to the tissue). If you have to stop at any point in the dehydration process 70% ethanol is best.
  2. Continue dehydration with two more changes of cold 100% ethanol (30 minutes per change).
  3. Follow cold 100% ethanol with one 30 minute change of cold (1:1) 100% ethanol : propylene oxide. Use propylene oxide in the fume hood.
  4. Complete dehydration with three changes of propylene oxide in the refrigerator, 30 minutes per change. Bring the tissue slowly to room temperature.
D. Infiltration
  1. Infiltrate tissue using the following concentrations of Spurr's resin, approximately 2-4 hours per change or longer: (1:2) Spurr's resin : propylene oxide, (2:1) Spurr's resin : propylene oxide. Refractory material may take up to one day per change.
  2. Use two changes of 100% Spurr's resin (2 hours or usually more per change).
  3. Embed in fresh Spurr's resin (less than one day old). Polymerize for 6 to 8 hours at 70° C.