Preparing Sections:

1. Paraffin sections mounted on slides, air dry overnight, and then dry in 50 C oven for 1 hour. The thickness of section should at least 5 um, and 10 um section is preferred.
2. Deparaffinize sections by placing slides in three changes of xylene or xylene substitute, 10 min each.
3. Re-hydrate sections through a series of alcohols (100% - 3 changes 5 min each, 95% - 1 min and 70% 1 min) to water.

Re-Fixation:

    The following steps (step 4 through step 14) can be performed in small plastic slide mailer.

4. Re-fix sections with 4% paraformaldehyde and 1% glutaraldehyde in 0.1M PB (phosphate buffer) pH 7.2 for 1-2 hours.
5. Rinse in 0.1M PB and incubate in 8% (0.2M) sucrose in 0.1M PB for 2x15 min or overnight.

Post-Fixation:

6. Post-fix in 1% osmium tetroxide in 0.1 M PB for 1 hour.
7. Rinse with 0.1M PB briefly.

Dehydration:

8. 50% ethanol  15min
9. 70% Ethanol  15min
10. 95% Ethanol  15min
11. 100% Ethanol  2x15min
12. 100% Propylene oxide  2x15min
13. 1:1 EMbed-812:Propylene Oxide (P.O.) for 1-2 hours.
14. 2:1 EMbed-812:P.O. for overnight in dessicator.

Embedding:

15. Drain off 2:1 EMbed-812:P.O. and place slides on a flat surface. Put a few drops of EMbed-812 on the sections and incubate for 1 hour, drain and change fresh EMbed-812 and incubate for another 1 hour. Then use one of the following methods to perform embedding.
16. Method 1: Use a flat pre-hardened block that you have previously made in a flat end beam capsule. Use the flat end of this block and place it on top of the section, being careful not to introduce air bubbles. Bake in 60 C oven for overnight (about 16 hours) or longer. Be sure to place the slide on a flat surface, otherwise the pre-hardened block may move and you may miss the section on the slide.
17. Method 2: Place slide on top of an Aclar Embedding Film (EMS Cat# 50425-10), make sure the size of the film is larger than the slide. Then cut off another film (size should be a little bigger than the slide), place it on top of the slide from one end to the other, being careful not to introduce air bubbles. Bake in 60 C oven for overnight (about 16 hours) or longer.

Detaching EMbed Block from Slide:

18. If you used Method 1 above: Take the slide and Epon block out of oven and let it cool down. Cut a rectangle or circle shape surrounding the EMbed block using razor blade (to make detachment easier!). Then immerse slide in liquid nitrogen for about 30 seconds to 1 minute, and this will detach the EMbed block together with section from the slide.
19. If you used Method 2 above: Take the slide out of oven and let it cool down. Peel off the Aclar Embedding Film from both sides of the slide. Cut a rectangle or circle shape surrounding the section using razor blade (to make detachment easier!). Then immerse slide in liquid nitrogen for about 30 seconds to 1 minute, and this will detach the section from the slide. Observe the “EMbed section”, choose the region of interest (ROI), cut a piece of ROI and glue it (using Tissue Adhesive - Permabond 910 from EMS, Cat# 72590) onto a flat end EMbed block, air dry for 30 minutes or overnight.

Sectioning:

20. Trim the block, and cut semithin (thick) sections at 0.5~1 um, be careful to line up the block surface perfectly and cut only one or two thick sections and no more, or you will lose the entire section if you used 5um sections. Transfer the thick sections to a drop of water (this will flatten sections to avoid wrinkles - very important!!!) on slide and bake it on a slide warmer or lamp for a few minutes until the drop of water is gone, and stain with toluidine blue for 2 min, rinse in tap water.
21. Observe under microscope for guidance of trimming and cutting ultrathin sections.
22. Ultrathin sections at 60-90 nm thick (bright yellow color-good, silver color-too thin, blue/purple color-too thick) and collect sections onto grids. Dry sections for a few hours before staining.

Staining:

Stain grids with uranyl acetate for 15 minutes and lead citrate for 5 minutes. Allow the grids to dry. Observe under electron microscope.

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 at 4 C. 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 C. 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.