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
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