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