Fixation:

1.   Fix 1 mm tissue blocks in 4% formaldehyde and 1% glutaraldehyde in 0.1 M PB (pH 7.4) for at least 2 hours to overnight.

2.   Immerse in 8% (0.2M) sucrose in 0.1 M PB 3x15min or overnight.

3.   Post-fix in 1% osmium tetroxide in 0.1 M PB 1 hour.

Dehydration:

4.   50% ethanol  15min

5.   70% Ethanol  15min

6.   95% Ethanol  15min

7.   100% Ethanol  2x15min

8.   100% Propylene oxide  2x15min

9.   1:1 EMBed 812 and Propylene Oxide for1-2 hour.

10. 2:1 EMBed 812:Propylene Oxide overnight in dessicator with top off.

Embedding:

11. Embed in beam capsules.

12. Bake in 60 C oven for 48 hours.

Sectioning:

13. Cut thick sections (0.5-1.0 um), and use a steel loop to transfer the sections to a drop of water (this will flatten sections to avoid wrinkles - very important!!!) on slide and dry it on slide warmer or lamp. Toluidine blue stain for 2-5 min.

14. Observe sections under microscope for precise location to cut for ultrathin sections

15. Ultrathin sections at 60-90 nm thick (silver-yellow color) and collect sections onto grids. Dry sections overnight before staining.

Staining:

16. Stain grids with uranyl acetate for 15 minutes and lead citrate for 5 minutes.

17. Observe under electron microscope.

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 at 4 ºC. This solution is stable for at least 6 months at 4 C.

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.