Procedure:

1.   Sectioning: Vibratome sections at 50 um thick.

2.   Collect sections in 0.1M PB.

3.   Pretreatment: treat sections with 1% sodium borohydride solution for 30 minutes.

4.   Rinse many times in 0.1M PB to remove bubbles.

5.   Rinse in PBS for 3x5min

6.   Serum Blocking: incubate in blocking buffer for 30 minutes.

7.   Primary Antibody: incubate sections with primary antibody (appropriate diluted in blocking buffer) overnight at room temperature.

8.   Rinse in PBS for 3x5min.

9.   Washing Buffer: incubate in washing buffer for 30 minutes.

10. Secondary Antibody: incubate sections in gold-conjugated secondary antibody diluted 1:50 in washing buffer for 3 hours.

11. Rinse 4x5 min in washing buffer and then 4x5 min in PBS.

12. Post-fixation1: fix sections with 2% glutaraldehyde in PBS for 10 minutes.

13. Rinse 4x5 minutes in PBS.

14. Silver enhancement:

·     Rinse in 0.2M citrate buffer 3x2min.

·     Incubate in silver enhancement solution for 3-9 minutes for EM and 6-12 minutes for LM (If room temperature is very low, a longer time may be needed such as 20-30 min).

·     Rinse in 0.2M citrate buffer 2x5min

·     Rinse in 0.1 M PB 4x5 min

15. Post-fixation2: fix sections with 1% OsO4 in 0.1M PB for 1 hour.

16. Rinse in 0.1M PB for 3x5min.

17. Dehydration: dehydrate in 50%, 70% and 95% ethanol for 10 minutes each.

18. 100% ethanol 2x15 min.

19. Propylene oxide 2x15 min.

20. Incubate sections in 1:1 mixture of Embed-812 & propylene oxide for overnight.

21. Transfer sections to straight Embed-812 for 2 hours.

22. Embedding: flat-embedding, and bake in 62 C oven for 48 hours.

23. Sectioning: choose the region of interest, cut off and glue on blank flat-end beam capsule using super glue. Bake to dry for 30 minutes to 1 hour. Trim and cut ultrathin sections at 60-90 nm.

24. Contrast Staining: stain sections with uranyl acetate for 15 minutes and lead citrate for 5 minutes.

27. Observation.

Solutions and Reagents:

0.2M Phosphate Buffer (0.2M PB):

To prepare 1 liter,

Na₂HPO_4, -------------------- 21.8 g

NaH₂PO₄ --------------------- 6.4 g

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

Mix to dissolve and adjust pH to 7.4

0.1M Phosphate Buffer (0.1M PB):

To prepare 1 liter,

0.2M PB --------------------- 500 ml

Distilled water -------------- 500 ml

10X Phosphate Buffered Saline (PBS):

To prepare 1 liter,

Na₂HPO₄ --------------------- 10.9 g

NaH₂PO₄ --------------------- 3.2 g

NaCl --------------------------- 90 g

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

Mix to dissolve and adjust pH to 7.4

0.2M Citric Acid:

To prepare 100 ml,

Citric acid (monohydrate) ------ 4.2 g

Distilled water -------------------- 100 ml

0.2M Sodium Citrate Buffer (fresh):

To prepare 100 ml,

Sodium citrate (dihydrate) ------ 5.88 g

Distilled water --------------------- 100 ml

Adjust pH to 7.4 with 0.2M citric acid (keeps refrigerated).

Fixative (4% Paraformaldehyde and 0.5% Glutaraldehyde in 0.1M PB):

To prepare 1 liter,

Paraformaldedyde -------------- 40 g

0.1M PB --------------------------- 1000 ml

Heat to 65 C. Add a few drops of 1N NaOH to clear the solution. Stir to dissolve. Filter the solution and add 10 ml of 50% glutaraldehyde.

Post-fixative (1% Osmium Tetroxide in 0.1M PB):

To prepare 20 ml,

4% osmium tetroxide ---------- 5 ml

0.2M PB --------------------------- 5 ml

0.1M PB -------------------------- 10 ml

2% Glutaraldehyde in PBS:

To prepare 100 ml,

50% glutaraldyhyde ------------ 4 ml

PBS -------------------------------- 100 ml

1% Sodium Borohydrite Solution in 0.1M PB:

To prepare 100 ml,

Sodium borohydrite -------------- 1 g

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

Stir to dissolve.

Blocking Buffer (1% BSA, 3% NGS, 0.04% Triton in PBS):

To prepare 100 ml,

Triton X-100 ----------------------- 0.04 ml

BSA ---------------------------------- 1 g

Normal goat serum -------------- 3 ml

PBS ---------------------------------- 100 ml

Stir to dissolve.

Washing Buffer (0.8% BSA, 0.1% Fish Gelatin in PBS).

To prepare 100 ml,

BSA ---------------------------------- 0.8 g

Fish gelatin ------------------------ 0.1 ml

PBS --------------------------------- 100 ml

Secondary Antibody

1nm gold conjugated secondary antibody from Amersham. Dilute the antibody 1:50 in washing buffer.

Silver Enhancement Solution:

Kit from Amersham and prepare solution according to the instruction provided.

Embed-812:

Embed-812 Kit from EMS containing Embed-812, DDSA, NMA, and DMP-30. Make Embed-812 according to instruction provided with the kit.

1:1 Mixture of Embed-812 & Propylene Oxide:

To prepare 20 ml,

Embed-812 ------------------------- 10 ml

Propylene oxide ------------------- 10 ml

5% Uranyl Acetate Solution:

To prepare 50 ml,

Uranyl acetate --------------------------2.5 g

Distilled water -------------------------- 50 ml

Cover with foil and stir overnight.

Add 10 drops of glacial acetic acid and store solution in 4 C for 3 months.

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.

Epon 812:

Final Volume	EM bed-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.