Cryo EM Tissue Preparation

1. Fix tissue mildly. Trim into small (<1mmsq) blocks.

2. Infuse tissue with a cryoprotectant for at least one hour (PVP/ sucrose).

3. Mount onto cryo pins, and freeze rapidly in liquid nitrogen.

4. Store pins in cryo vials in liquid nitrogen dewar.

1. Cut ultra thin sections (50-80nm)

2. Pick up section with sucrose drop.

3. Place on formvar / carbon coated Nickel grid.

4. Wash off excess sucrose.

5. Block for nonspecific binding with 10% FCS 1 hour.

6. Place grid section side down on 10ul drop of diluted antibody. for several hours / overnight.

7. Wash with blocking buffer.

8. Incubate grids section side down on secondary antibody, (5-10nm IgG colloidal gold).

9. Wash blocking buffer/ wash water.

10. Stain 20 mins with neutral UA.

11. Wash with water.

12. Stain and embed section in a monolayer on PVA / Methyl cellulose.

Let dry and scope.