Fixation:

1. Fix cell suspension or free cells in 4% formaldehyde and 1% glutaraldehyde in 0.1 M PB (pH 7.4) by mixing equal volume of fixative and cell suspension.
2. Transfer cells to centrifuge tube and spin for 10 minutes. A nice, tight pellet will be formed (Re-spin if necessary during processing). Carefully pipette off fixative. Add fresh fixative for at least 2 hours or overnight.
3. Pipette off fixative. Replace with 8% (0.2M) sucrose in 0.1 M PB 3x15 minutes or overnight at 4 C (Samples can be kept in sucrose for a long time).
4. Post-fix with 1% OsO₄ in 0.1 M PB for 1 hour.
5. Pipette off OsO₄, Rinse in 0.1 M PB 3x10 minutes.

Dehydration:

1. 50% ethanol  15min
2. 70% Ethanol  15min
3. 95% Ethanol  15min
4. 100% Ethanol  2x15min
5. 100% Propylene oxide  2x15min
6. 1:1 EMBed 812 and Propylene Oxide for1-2 hour.
7. 2:1 EMBed 812:Propylene Oxide overnight in dessicator with top off.

Embedding:

Embed in Beam capsules, and bake in 60 ºC oven for 48 hours.

Sectioning:

Semithin (thick section, 0.5-1 um) and toluidine blue staining. Observe the thick section, and trim EM block further if needed. Then ultrathin sectioning, and collected on grids.

Staining:

Staining grids with uranyl acetate (see reagent below) for 15 minutes, rinse with distilled water and then staining with lead citrate (see reagent below) for 3-5 minutes, rinse with distilled water.

Solutions and Reagents:

0.2M PB, pH 7.4:

Na₂HPO4 ------------------------ 21.8 g

NaH₂PO4 ------------------------ 6.4 g

Distilled water ----------------- 1000 ml

4F1G Fixative (4% Formaldehyde & 1% Glutaraldehyde in 0.1 M PB, pH 7.4):

0.1M PB, pH 7.4 --------------- 88 ml

37-40% Formaldehyde ----- 10 ml

50% Glutaraldehyde --------- 2 ml  

8% (0.2M) Sucrose in 0.1M PB:

Sucrose ------------------------- 8 g

0.1 M PB ------------------------ 100 ml

1% Osmium in 0.1M PB:

2% Osmium -------------------- 5 ml

0.2M PB, pH7.4 --------------- 5 ml

5% Uranyl Acetate Solution:

To prepare 50 ml, add 2.5 g of uranyl acetate to 50 ml of distilled water. Cover with foil and stir overnight. Add 10 drops of glacial acetic acid. Store solution in 4 ºC.

Reynold’s Lead Citrate Solution:

To prepare 50 ml, add chemicals in distilled water in following order

Lead nitrate ------------------------------ 1.33 g

Sodium citrate, dihydrate -------------- 1.76 g (solution becomes cloudy when sodium citrate is added)

1N NaOH ----------------------------------- 5 ml  (solution becomes clear when NaOH is added)

Distilled water ----------------------------- 30 ml

Stir for 10 minutes to dissolve and add additional 15 ml of distilled water. Store solution for 3-6 months at 4 ºC. Note: the mount of NaOH is very important. Too much will make solution cloudy.

EMBed 812:

Final Volume	EMBed-812	DDSA	NMA	DMP-30 (2%)
20.91 ml	10 ml	4.5 ml	6 ml	0.41 ml
31.37 ml	15 ml	6.75 ml	9 ml	0.62 ml
41.82 ml	20 ml	9 ml	12 ml	0.82 ml
52.28 ml	25 ml	11.25 ml	15 ml	1.03 ml

        Mix all four components in plastic beaker and stir with wood stick.