TABLE 1.
Drosophila sample preparation procedures and results.
| Methods | ||||
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| Procedure and results | 1. HPF-FS | 2. PLT-LTS | 3. PLT-LTS heavy metal enhancement | 4. PLT-LTS progressive heavy metal enhancement |
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| Tissue dissection Pre-fixation |
200 μm Vibratome slices | Brain tissue dissect out with metal collar | CNS dissect out with metal collar | Brain dissect out with/out metal collar |
| Pre-fixation | 2.5%GA + 2.5% PFA, RT, 15 min | 2.5%GA + 2.5% PFA, RT, 2 h | 2.5%GA + 2.5% PFA, RT, 2 h | 2.5%GA + 2.5% PFA, RT, 2 h |
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| Post-fixation Heavy metal enhancement |
HPF cryo-fixation | 0.5% OsO4, 30 min, 4°C, W 0.5% UA, 30 min, 0°C, W |
0.5% OsO4, 40 min, 4°C, W no wash, change to 0.8% K ferrocyanide, 2 h, 0°C + 0.5% UA, 30 min, 0°C; W lead aspartate, 4°C overnight; or 4 h RT W 1% OsO4, 20 min, 0°C |
1% OsO4, 40 min, 4°C, W no wash, change to 1.5% K ferrocyanide, 1.5 h, 0°C + 30 min RT; W 1% Tch, 15 min, RT; W, 2% OsO4, 30 min, RT; W 0.5% UA, 30 min, RT, W; lead aspartate, 30 min, 55°C, and 1 h, RT, W |
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| en bloc staining and dehydration | FS: 95% acetone, 1% OsO4, 0.2% UA, 1% methanol, 38 h -90°C 14 h -20°C |
PLT: acetone, 1% OsO4, 0°C to -25°C LTS: 97% acetone with 1% OsO4 and 0.2% UA, 30 h, -25°C |
PLT: ethanol, 0°C to -25°C LTS: 96% ethanol with 1% OsO4 and 0.2% UA, 30 h, -25°C |
PLT: ethanol 0°C to -25°C LTS: 96% acetone with 0.2% UA, or 96% ethanol with 1% PTA 30 h, -25°C |
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| Infiltration Embedding |
Acetone Durcupan |
Propylene oxide Poly/Bed 812 |
Propylene oxide Poly/Bed 812 |
Acetone Durcupan |
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| Summary | ||||
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| Hot knife cutting | Not suitable | 20 μm slices | 25 μm slices on BSA en coating tissue | Not suitable |
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| FIB-SEM 8 nm imaging scan rate | 1.25 MHz | 1.25 MHz | 2.5 MHz | 10 MHz |
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| FIB-SEM 8 nm imaging speed | 20 × 103 μm3 per day | 20 × 103 μm3 per day | 40 × 103 μm3 per day | 150 × 103 μm3 per day |
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| FIB-SEM data collection | Medulla, lobula, lobula plate; α-lobe; antennal lobe |
Hemi-brain (central complex, mushroom body and more) | Central nervous system (CNS) | Medulla 1st instar larva CNS |
LTS, Low temperature staining; W, washing (3 × 10 min). Hot knife cutting properties improved by infiltration in propylene oxide not acetone, and Epon (Poly/Bed 812, Ted pella) instead of Durcupan.