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 and lead citrate. 17. 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) |