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)
- 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.
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.