TEM Immunogold Protocol (click here for an example image)

1. Incubate grids in 50mM NH4Cl for up to 30 minutes (anything fixed with aldehydes) or in 5% H2O2 (use with osmicated samples)

2. Wash grids in water and blot dry.

3. Incubate grids in 5% low/non fat Carnation Instant Milk in PBST buffer for 30 min.

4. Wash grids and blot dry.

5. Incubate grids on 1 O antibody dilutes 1/50 (or whatever is recommended) in PBST for 1-4 hours in 37C or overnight at 4C.

6. Wash grids in PBST for 30s and blot dry.

7. Incubate grids in 2 O antibody in PBST (1/50 or 1/100) for 30-60minutes at 37C.

8. Wash in PBST 3X for 30s

9. Wash in DH2O 3X for 30s

10. Blot dry and stain in UA/ Pb Citrate

Solutions

PBS Stock : 10nM Na2PO4, 500mM NaCl, 0.1% Tween 20

PBS : 25ml of   PBS Stock and add 225ml of dH20

PBST : add 0.1ml of Tween 20 to 100ml of PBS

Carnation Milk Block : add 2.5g or Carnation Instant Milk to 50 ml of PBST

1' antibody solution: 1/50- 10ul of antibody to 0.5ml PBST

                                    1/100-10ul of antibody   to 1ml PBST

2' antibody solution 1/50- 10ul of anti-rat or anti-mouse antibody to 0.5ml of PBST(AB labeled with 10-20nm gold particles)

NH4CL(50mM): 0.135g of NH4CL in 50ml of DH2O