Stains for Electron Microscopy

Methanolic Uranyl Acetate 
Add 1.25 g uranyl acetate to 25 mL of absolute methanol and sonicate until most is dissolved. Let stand overnight and store at 4OC. 
Aqueous Uranyl Acetate 
Add 5 g uranyl acetate to 100 mL water. Sonicate. Store at 4OC. 
Reynold's Lead Citrate 
	1.33 9 Pb(N03)2 (lead nitrate)
1.76 9 Na3(C6H307) .H20 (sodium citrate) 30 mL distilled water 
Mix in 50 mL volumetric flask, shake 1 min and let stand with intermittent shaking for 30 min. After 30 min, add 8.0 ml of 1 N NaOH (fresh) or, better yet, add 0.32 g NaOH directly (about 4 NaOH pellets). Dilute to 50 mL with distilled water. Final pH about 12. Stores at 4OC about 1 month. 

IMMUNOGOLD STAINING OF ULTRA-THIN SECTIONS 
1.	Collect sections on clean nickel grids. (Place grids in 40-50°C oven for 15-30 min prior to staining if there is a problem with sections floating away during staining procedure.) 
	Note: 	Carry out staining procedure by floating grids on reagent drops. 
Try not to allow grids to sink in reagent drop. Once hydrated (step 2) do not let sections to dry until step 11. 
2.	Hydrate sections in Tris buffered saline (TBS: 20 mM Tris-HCl, 0.9% NaCl) for 10 min. (For polyclonal AB use pH 7.2-7.5; for mAbs, use pH 8.0-8.2.) 
	3. 	Etch two minutes in 5% H202• 
	4. 	Wash grids in TBS (at least four changes, 5 min each). 
	5. 	Blocking: incubate grids 30 min in TBS-1% BSA. 
6.	Incubate overnight (4°C) in primary antibody appropriately diluted in blocking buffer (TBS-1% BSA). (Set up a moist chamber for this and secondary antibody incubations.) 
	7. 	Wash 5X (3-5 min each) in TBS-T (TBS containing 0.05% Tween-20). 
	8. 	Incubate in appropriately diluted secondary antibody 1 hr, RT. 
	9. 	Wash 2X (3-5 min each) in TBS-T. 
10.	Dip grids rapidly in TBS-T (100 dips) followed by 100 rapid dips in dH2O. 
11. Dry grids on clean filter paper. 
12. stain with uranyl acetate and lead citrate as for routine TEM 

3.	
4.	
5.	
INSTRUCTIONS FOR PIPET WASHER 
	1. 	Place pipets to be washed in basket, tips up, use 1 Alconox tablet or half of the small stainless beaker of dry power Alconox. 
	2. 	Place basket into washer.
	3. 	Hook washer up to faucet (See diagram, #1).
	4. 	Place washer so that waste water empties into sink (#2).
	5. 	Turn on hot water qentlv, at a rate of approximately 3 minutes fill time.
	6. 	Allow pipets to rinse at least ten times (about 30 min).
	7. 	Turn off water and disconnect washer.
8.	Rinse pipets two times in distilled water or nanopure water and place in oven to dry 
	9. 	Put dried pipets away or prepare for sterilization 

6.	
GEL DRYING PROCEDURE 
	1. 	Dry dessicant for at least 1 h in 60C oven. 
	2. 	Assemble drying apparatus as shown at left. 
	3. 	Place flask for cold trap in ice and sprinkle salt on top. 
	4. 	Soak gel (with agitation) in 2% glycerol for 30-60 min. 
5.	Position gel on filter paper (blotter paper) – or cellophane. See instruction booklet for details. 
	6. 	Turn on pump. Check to be sure silicon flap forms seal. 
7.	Set timer. Time required will vary with gel thickness (1.5 rom gel takes approximately 1.5 hr). 
	8. 	Allow pump to run 15-30 min after timer stops for cooldown. 
Normal arrangement for gel drying: 

5 	Silicon rubber flap 	(top) 		
4 	Mylar 			
3 	Gel and blotter paper 		
2 	Blotter paper 			
1 	Perforated steel support 	(bottom))