Citrus Systemic Pathogen Research

SSEM

 

(Note: Grids are coated on the dull side and that side is facing "up" on the parafilm) Next >

1. Float formvar/carbon coated grids on appropriately diluted antibody in 0.05 M Tris pH 8(we use CTV at 1/500) for 30 minutes to 1 hour.

2. Rinse lightly by placing briefly on a drop of 0.05 M Tris pH 8 and place on antigen source for 30 minutes to 1 hour.

3. Rinse by placing the grid on three consecutive drops of 0.05 M Tris pH 8 followed by three consecutive drops of distilled water.

4. ( Note: Do not disturb the solid residue on the bottom of the bottle.) Place grid onto drop of 5% Uranyl Acetate in 50% Ethanol for ~2 minutes.

5. Destain by dipping grid 15 to 20 times into 95% Ethanol and allow to air dry.  Place in grid holder and label grid holder slots.

SSEM with gold labeling

6. Float formvar/carbon coated grids on appropriately diluted antibody in 0.02 M Tris pH 8.2 for 30 minutes to 1 hour.

7. Rinse lightly by placing briefly on a drop of 0.02 M Tris and place on antigen source for 30 minutes to 1 hour.

8. Rinse by placing grid on three consecutive drops of 0.2 M Tris and 0.1% BSA, then place grid on a drop of Tris/BSA for 10 to 15 minutes.

9. Place grid on appropriately diluted antibody in Tris/BSA for 30 minutes.

10. Rinse by placing grid on three consecutive drops of Tris/BSA and place on drop of appropriately diluted gold labeled secondary antibody (depending on source of primary antibody) in Tris/BSA for 30 minutes.

11. Rinse by placing grid on two consecutive drops of Tris/BSA and then 1 drop of distilled water.

12. ( Note: Do not disturb the solid residue on the bottom of the bottle.) Place grid onto drop of 5% Uranyl Acetate in 50% Ethanol for ~2 minutes.

13. Destain by dipping grid 15 to 20 times into 95% Ethanol and allow to air dry.  Place in grid holder and label grid holder slots.