Sectioning:

10.    Semithin section and Toluidine blue stain for 2-5 min

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

12.    Ultrathin sections

Staining:

13.    Uranyl acetate and lead citrate

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 C.

Reynold’s Lead Citrate Solution:

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