Citrus Systemic Pathogen Research

LR White Embedding Protocol

 

Fixatives and buffers < Previous -- Next >

  • 0.1M PIPES

3.02 g PIPES in 100 ml distilled water.  Adjust pH to 7.2 with sodium hydroxide.  The solution will clear as the pH is raised.

  • 8% Formaldehyde

Heat 50 ml of distilled water and 4 g paraformaldehyde to 60C with stirring in a fume hood.  Add 1 N sodium hydroxide drop-wise until the solution clears.  Cool.

  • 0.2% Glutaraldehyde

Dilute EM grade 25% glutaraldehyde to 0.2% using 0.1 M PIPES

Procedure:

  • 1. Mix the 8% formaldehyde and 0.2% glutaraldehyde to make a solution of 4% formaldehyde and 0.1% glutaraldehyde mixture in 0.05 M PIPES.
  • 2. Dilute the 0.1 M PIPES with distilled water to a final concentration of 0.05 M PIPES.
  • 3. Fix tissue for 1 2 hours at room temperature or overnight at 4 C.
  • 4. Wash three times with 0.05 M PIPES for 15 minutes or more each wash.
  • 5. Store at 4 C.
  • 6. Dehydrate in a graded ethanol series (25%, 50%, 75%, 90%, 100%) for 15 minutes each step, then in 100% ethanol for 1 hour and then in 100% ethanol overnight at 4 C.
  • 7. Infiltrate with LR White for 15 minutes with 3:1 LR White/ethanol, 15 minutes with 2:1 LR White/ethanol, 15 minutes 1:1 LR White/ ethanol, 15 minutes LR White, 1 hour LR White and overnight with LR White at 4 C.
  • 8. Embed in gelatin capsules at ~ 62 C.

Gold labeling of sections

  • Collect sections on Nickel grids.
  • Etch with 0.4 M sodium meta-periodate for 2 to 5 minutes (NOTE: Etching time can be adjusted to optimize labeling).
  • Rinse on two drops of filtered, distilled water.
  • Incubate grid on a drop of 0.02 M Tris pH 8.2 and 0.1% BSA (Tris/BSA) for 10 minutes.
  • Place grid on drop of diluted antibody in Tris/BSA for 30 minutes at 37 C (NOTE: Antibody dilution can be adjusted to optimize labeling).
  • Wash by placing grid on 3 consecutive drops of Tris/BSA for 5 minutes each drop.
  • Place grid onto a drop of gold-labeled secondary antibody in Tris/BSA for 20 minutes at 37 C.
  • Wash by placing grid onto 3 consecutive drops of Tris/BSA for 5 minutes each drop, followed by 2 consecutive drops of filtered, distilled water.
  • Stain grid by placing it on a drop of 7% aqueous uranyl acetate for 30 minutes, place grid on a drop of filtered, distilled water to remove excess stain and place on a drop of Reynolds lead citrate (in a petri dish with sodium hydroxide pellets) for 5 minutes, followed by a wash with 0.2 M sodium hydroxide followed by a wash with distilled water.  Air dry.