Table 1 Detailed comparison of staining steps in employed staining protocols
From: A carbon nanotube tape for serial-section electron microscopy of brain ultrastructure
Protocol | BROPA (Mikula 2015) | Hua (2015) | mHMS | TO | TOLA |
|---|---|---|---|---|---|
Tissue size | Whole mouse brain | 1 mm ϕ × 2 mm block | 50 µm-thick section | 50 µm-thick section | 50 µm-thick section |
Step 1 | 40 mM OsO4, 35 mM K4[Fe(CN)6], 2.5 M formamide, 0.1 M cacodylate, pH 7.4 | 2% OsO4, 0.15 M cacodylate, pH 7.4 | 2% OsO4, 1.5% potassium ferrocyanide, 0.1 M PB, pH 7.4 | 1% OsO4, 1.5% potassium ferrocyanide, 0.1 M PB, pH 7.4 | 1% or 2% OsO4, 1.5% potassium ferrocyanide, 0.1 M PB, pH 7.4 |
Time @ temp. 1 | 96 h @ RT | 1.5h @ RT | 1h @ 4 °C | 1h @ 4 °C | 1h @ 4 °C |
Wash 1 | ⇓ | ⇓ | 10 min wash in water ×3 | 10 min wash in 0.1 M PB ×3 | 10 min wash in 0.1 M PB ×3 |
Step 2 | 40 mM OsO4, 0.1 M cacodylate, pH 7.4 | 2.5% potassium ferrocyanide, 0.15 M cacodylate, pH 7.4 | ⇓ | ⇓ | ⇓ |
Time @ temp. 2 | 96 h @ RT | 1.5h @ RT | ⇓ | ⇓ | ⇓ |
Wash 2 | 4 h wash in 0.1 M cacodylate | 30 min wash in water ×2 | ⇓ | ⇓ | ⇓ |
Step 3 | 320 mM pyrogallol unbuffered, pH 4.1 | 1% TCH unbuffered | 1% TCH unbuffered | ⇓ | ⇓ |
Time @ temp. 3 | 72 h @ RT | 45 min @ RT | 20 min @ RT | ⇓ | ⇓ |
Wash 3 | 4 h wash in 0.1 M cacodylate | 30 min wash in water ×2 | 10 min wash in water ×3 | ⇓ | ⇓ |
Step 4 | 40 mM OsO4 unbuffered | 2% OsO4 unbuffered | 2% OsO4 unbuffered | 1% OsO4, 0.1 M PB, pH 7.4 | 1% or 2% OsO4, 0.1 M PB, pH 7.4 |
Time @ temp. 4 | 96h @ RT | 1.5h @ RT | 0.5h @ RT | 1h @ RT | 1h @ RT |
Wash 4 | 4h wash in water | 30 min wash in water ×2 | 10 min wash in water ×3 | 10 min wash in water ×3 | 10 min wash in water ×3 |
Step 5 | ⇓ | 1% uranyl acetate unbuffered | 1% uranyl acetate unbuffered | 1% uranyl acetate unbuffered | 1% uranyl acetate unbuffered |
Time @ temp. 5 | ⇓ | overnight @ 4 °C 2h @ 50 °C | overnight @ RT | 40 min @ RT | overnight @ RT |
Wash 5 | ⇓ | 30 min wash in water ×2 | 10 min wash in water x3 | 10 min wash in water x3 | 10 min wash in water x3 |
Step 6 | ⇓ | Lead aspartate, pH 5.0 | Lead aspartate, pH 5.5 | ⇓ | Lead aspartate, pH 5.5 |
Time @ temp. 6 | ⇓ | 2h @ 50 °C | 30 min @ 60 °C | 1h @ 60 °C | |
Wash 6 | ⇓ | 30 min wash in water ×2 | 10 min wash in water ×3 | 10 min wash in water ×3 | 10 min wash in water ×3 |
Step 7 | Dehydration, infiltration, embedding and hardening | Dehydration, infiltration, embedding and hardening | Dehydration, infiltration, embedding and hardening | Dehydration, infiltration, embedding and hardening | Dehydration, infiltration, embedding and hardening |
Figure panels | Fig. 7b–e | Figs. 1–3, 5a, g, m, s, Sup Fig. 1, 2, 4, 5, 6d, e, 7a-d, 8, 9 | Figs. 4f–k, 6b–d, h–j, n-p, t-v, x |
