Citrus Systemic Pathogen Research

Immunogold Labeling of Bacteria on Leaf Surfaces

 

Reagents < Previous

2 mM borate pH 9.2

  • 0.1 M potassium carbonate
  • 0.1 M Tris-HCl
  • 2.5% polyethylene glycol (MW 20,000)

TBS-BSA pH 8.3

  • 0.01M Tris
  • 0.15 M NaCl
  • 0.02% NaN3
  • 0.25% BSA
  • 0.1 M cacodylate pH 7.4

Attachment of colloidal gold to IgG for direct immunolabeling

IgG is dialyzed in 2 mM borate pH 9.2

The pH of 30 - 40 nm gold is adjusted to ~9.5 with 0.1 M potassium carbonate.  13.95 ml of  the colloidal gold and 1.05 ml of 0.1 M Tris-HCl pH 9.5 is placed in a sterile, siliconized beaker.  The dialyzed IgG was added slowly and stirred for 15 minutes at room temperature.  240 m l of 2.5% polyethylene glycol  was added to quench the IgG-gold absorbtion.

The pH was adjusted to 8.2 with HCl and bovine serum albumin (BSA) was added to a final concentration of 0.25%. 

Centrifuge at 12,000 x g for 5 minutes wash the red mobile pellet with TBS-BSA and repeat centrifugation.  Resuspend the pellet in 1.5 ml of  TBS-BSA and store at 4 C.

Fixation and labeling

  • Fix leaf discs in 1.5 % glutaraldehyde in 0.1 M cacodylate with 1,500 ppm ruthenium red pH 7.4 overnight at 4 C.
  • Wash discs three times in 0.1 M cacodylate with 5% sucrose pH 7.4.
  • Incubate leaf discs in 0.1 ml of TBS-BSA with 1% normal goat serum for 15 minutes.
  • Blot discs and place on 0.1 ml of gold-IgG complex diluted 1/10 in TBS-BSA overnight at 4 C.  (Direct labeling)

                                                            OR

  • Blot discs and place on 0.1 ml of specific IgG diluted 1/10 in TBS-BSA overnight at 4 C. 
  • Rinse 3 times for 5 minutes each rinse on 0.1 ml drops of TBS-BSA and place on a 0.1 ml drop of gold-goat anti-(whatever animal the primary antibody was developed in) diluted 1/10 in TBS-BSA overnight at 4 C.  (Indirect)
  • Rinse three times for 5 minutes each rinse on 0.1 ml drops of filtered double distilled water.
  • Discs were dehydrated in a 10 - 100% ethanol series, critical point dried, mounted on carbon planchets, given 3 coats of carbon and examined using a scanning electron microscope with a backscattered electron detector.

Reference:

Davis, C. L. and R. H. Brlansky.  1991.  Use of Immunogold Labelling with Scanning Electron Microscopy to Identify Phytopathogenic Bacteria on Leaf Surfaces.  Appl. Environ. Microbiol.  57:3052-3055.