Antibody Reactions for Immunogold Electron Microscopy

1. If sections were collected on the grids the same day that the antibody reactions are to be done, bake the grids at 55°C for 2 minutes.
2. Moisten the sponge in the humidity chamber, and place a fresh sheet of parafilm inside.
3. Place the grids in drops (~50ml) of blocker (PBST + 2% BSA) on the parafilm for 15-30 minutes.
4. Dilute primary antibody in blocker, and preadsorb to acetone powders when appropriate. (Add acetone powders, finger vortex, and leave at room temperature for 20-30 minutes. Spin down acetone powders at top speed and remove diluted/preadsorbed antibody). Place drops (~10-15ml) of primary antibody on parafilm in humidity chamber.
5. Transfer grids from blocker to primary antibody, after gently drying each with a kimwipe. Incubate grids in primary antibody for 1 hour.
6. Rinse the porcelain dishes with ddH2O, and swipe each well with a kimwipe to remove dust, etc. Add 0.3-0.5ml PBST to each well.
7. Transfer grids from primary antibody to PBST (drying in between), and shake for 2-5 minutes to wash. Wash each grid 3 times.
8. Transfer grids to drops of blocker in the humidity chamber, and incubate for 15-30 minutes.
9. Dilute secondary antibody (goat a-rabbit IgG-gold) 1:30 in blocker, and distribute into drops (10-15ml) on parafilm. Transfer grids from blocker to secondary antibody (drying in between). Incubate in secondary for 1 hour.
10. Rinse wells in porcelain dish as previously described, and add new PBST. Wash grids twice with PBST and once with filtered ddH2O.
11. Stain grids for 20-30 seconds in 1% uranyl acetate. Wash five times with filtered ddH2O.
12. Cross your fingers...