| Table of Contents |
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Abbreviations
- ACSF = artificial cerebrospinal fluid; composition in mM -- 116.4 NaCl, 5.4 KCl, 3.2 CaCl2 , 1.6 MgSO4 , 26.2 NaHCO3 , 1.0 NaH2PO4 , and 10 D-glucose
- EtOH = ethanol
- KRC = Krebs–Ringer Carbicarb buffer; composition in mM: 2.0 CaCl2, 11.0 d-glucose, 4.7 KCl, 4.0 MgSO4, 118 NaCl, 12.5 Na2CO3, 12.5 NaHCO3; pH 7.4; osmolality, 300–330 mmol/kg
- MW = microwave
- PND = postnatal day
- PO = propylene oxide
- RT = room temperature
Protocols
| Sorra and Harris, (1998)Bourne | Kirov et al. ( | 20071999) | Bourne et al. (2007) | Harris Bourne et al. (2015)Kirov et al. (19992007) | Bourne and Harris (2011) | dentate gyrus | SLM | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 3DEM Series | UGXI, NQDBP, ZSGCZ | HZKDG | GWPMS (also HCSBR and HWVKW?) | "Spine" (aka K24), "Oblique" (aka Rat34CA1BS12 1-91), "Apical" (aka LRHXG, volumejosef, or Rat34CA1BS12 101-294) | UGXI, NQDBP, ZSGCZ | WRPV | HZKDG | GWPMS (also HCSBR and HWVKW?) | Rat85LM | FHLTD, FWNGV, FPNCT, XRZCT | |||||
| Animals | Rat34 | Kari68, Kari69 | Rat84R | JB072 | JB031 (also JB037 and JB047?) | Rat34 | Rat84R | Rat85 | JB023, JB024, | BLE5, LED50, LED56, LE102, LE108, LE113 | MK01, LE108 | ||||
| Species | Rat | Rat | Rat | Rat | Rat | Rat | Rat | Rat | Rat | ||||||
| Strain | Long Evans | Long Evans | Long Evans | Long Evans | Long Evans | Long Evans | Long Evans | Long Evans | Long Evans | ||||||
| Age (PND) | 77 | 50-60 | 68 | 65 | 56-64 | 77 | 68 | 68 | 60 (JB023); 61 (JB024) | 185 (BLE5); 179 (LED50); 121 (LED56); 146 (LE102); 170 (LE108); 150 (LE113) | 162 (MK01); 170 (LE108); | ||||
| Sex | male | male | male | male | male | male | male | male | male | ||||||
| body weight (g) | 310 | 279 (Kari68); 236 (Kari69) | 411 | 333 | 263 (JB031); 361 (JB037); 365 (JB047)310 | 411 | 337 | 323 (JB023); 319 (JB024); | 490 (BLE5); 623 (LED50); 448 (LED56); 540 (LE102); 648 (LE108); 490 (LE113) | 648 (LE108); | |||||
| anesthesia | rapid decapitation | halothane | halothane | pentobarbital 80mg/kg (ip) | rapid decapitation | pentobarbital 80mg/kg (ip) | halothane | halothane | pentobarbital 80mg/kg (ip) | halothane | halothane | halothane | |||
| slice preparationdissection | N/A | dissection in ice-cold ACSF; recovered in ACSF at 30-31°C for at least 1 hr before recordings | N/A | dissection at RT; recovered in ACSF at 31°C before electrophysiology recordings | dissection at RT; recovered in ACSF at 31°C before electrophysiology recordings | N/A | N/A | N/A | dissection at RT; recovered in ACSF at 31°C before electrophysiology recordings | N/A | N/A | ||||
| total time (min) in recording chamber | N/A | 340 (Kari68); 270 (Kari69) | N/A | 260 | 285 (JB031); 235 (JB037); 285 (JB047) | N/A | N/A | N/A | 315 (JB023); 360 (JB024); | N/A | N/A | ||||
| tissue fixation | Perfused w/ 2% paraformaldehyde, 6% 2.5% glutaraldehyde, and 2 mM CaCl2 in 0.1 M sodium cacodylate buffer during 8 s MW, pH 7.35, 37°C, 4 psi pressure | 2% paraformaldehyde, 6% glutaraldehyde, in 0.1 M sodium cacodylate buffer with 1 and 2 mM CaCl2 and 2 mM MgCl2 during 8-20 s MW2% paraformaldehyde, 6% glutaraldehyde, in 0.1 M sodium cacodylate buffer with 1 mM CaCl2 and 2 mM MgCl2 during 8 -20 s MW | Perfused w/ 2% paraformaldehyde, 2.5% 6% glutaraldehyde, 2 mM CaCl2, 4 mM MgSO4 in 0.1 M sodium cacodylate buffer, pH 7.354, 37°C, 4 psi pressurePerfused w/ | 2% paraformaldehyde, 6% glutaraldehyde, 2 in 0.1 M sodium cacodylate buffer with 1 mM CaCl2 and 2 , 4 mM MgSO4 mM MgCl2 during 8-20 s MW | 2% paraformaldehyde, 6% glutaraldehyde, in 0.1 M cacodylate buffer, pH 7.4, 37°C, 4 psi pressuresodium cacodylate buffer with 1 mM CaCl2 and 2 mM MgCl2 during 8-20 s MW | Perfused w/ 2% paraformaldehyde, 6% glutaraldehyde, 2 mM CaCl2, 4 mM MgSO4 in 0.1 M cacodylate buffer, pH 7.4, 37°C, 4 psi pressure | 2% paraformaldehyde, 6% glutaraldehyde, in 0.1 M sodium cacodylate buffer with 2 mM CaCl2, 4 mM MgSO4 during 8-20 s MW | Perfused w/ KRC, followed by 2% formaldehyde, 2.5% glutaraldehyde, 2 mM CaCl2, 4 mM MgSO4 in 0.1 M cacodylate buffer, pH 7.35, 37°C, 120 mmHg pressure | Perfused w/ KRC, followed by 2% formaldehyde, 2.5% glutaraldehyde, 2 mM CaCl2, 4 mM MgSO4 in 0.1 M cacodylate buffer, pH 7.35, 37°C, 120 mmHg pressure | ||||||
| 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | |||||||
| reduced osmium | 1.5% K4Fe(CN)6, 1 % OsO4 in 0.1 M sodium cacodylate buffer, 1 hr | 1.5% K4Fe(CN)6, 1 % OsO4 in 0.1 M sodium cacodylate buffer, 1 hr | 1.5% K4Fe(CN)6, and 1% OsO4 in 0.1 M sodium cacodylate buffer, MW (175 W) under vacuum, 1 min on - 1 min off - 1 min on. Cool cool to below 15°C and repeatMW 2.5 min. | 1.5% K4Fe(CN)6, 1 % OsO4 in 0.1 M sodium cacodylate buffer, 1 hr | 1.5% K4Fe(CN)6 and , 1% OsO4 in 0.1 M sodium cacodylate buffer, cool , MW (175 W) under vacuum, 1 min on - 1 min off - 1 min on. Cool to below 15°C and MW 2.5 minrepeat. | 1.5% K4Fe(CN)6 and 1% OsO4 in 0.1 M sodium cacodylate buffer, cool to below 15°C and MW 2.5 min. | 1.5% K4Fe(CN)6, 1 % OsO4 in 0.1 M sodium cacodylate buffer, 10 min. | 1.5% K4Fe(CN)6, 1 % OsO4 in 0.1 M sodium cacodylate buffer, 5 min. | 1.5% K4Fe(CN)6, 1 % OsO4 in 0.1 M sodium cacodylate buffer, 5 min. | ||||||
| 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | |||||||
| osmium | 1% OsO4, 1 hr | 1% OsO4, 1 hr | 1% OsO4 in 0.1M 1 M sodium cacodylate buffer, MW (175 W) under vacuum. 1 min on - 1 min off - 1 min on. Cool cool to below 15°C and repeatMW 2.5 min. | 1% OsO4, 1 hr | 1% OsO4 in 0.1M sodium cacodylate buffer, MW (175 W) under vacuum. 1 M sodium cacodylate buffer, cool 1 min on - 1 min off - 1 min on. Cool to below 15°C and MW 2repeat.5 min. | 1% OsO4 in 0.1 M sodium cacodylate buffer, cool to below 15°C and MW 2.5 min. | 1% OsO4 in 0.1M sodium cacodylate buffer, MW (175 W) under vacuum. 1 min on - 1 min off - 1 min on. Cool to 20°C and repeat. | 1% OsO4 in 0.1M sodium cacodylate buffer, MW (175 W) under vacuum. 1 min on - 1 min off - 1 min on. Cool to 18°C and repeat. | 1% OsO4 in 0.1M sodium cacodylate buffer, MW (175 W) under vacuum. 1 min on - 1 min off - 1 min on. Cool to 18°C and repeat. | ||||||
| 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | 0.1 M sodium cacodylate buffer washes | |||||||
| ddH2O rinses | ddH2O rinses | ddH2O rinses | ddH2O rinses | ddH2O rinses | ddH2O rinses | ddH2O rinses | ddH2O rinses | ||||||||
| dehydration & UA | 30% EtOH, 10 min | 50% EtOH, 10 min | 1% UA (aq), cooled on ice, MW 2.5 min | 50% EtOH with 1% UA, 10 min | 50% EtOH with 1% UA, MW (250W) without vacuum, 40 s | 30% EtOH, 10 min | 1% UA (aq), cooled on ice, MW 2.5 min | 1% UA (aq), cooled on ice, MW 2.5 min | 50% EtOH with 1% UA, MW (250W) without vacuum, 40 s | 50% EtOH with 1% UA, MW (250W) without vacuum, 40 s | 50% EtOH with 1% UA, MW (250W) without vacuum, 40 s | ||||
| 50% EtOH, 10min | 70% EtOH with 1% UA, 1 hr | ddH2O rinses × 2 | 70% EtOH with 1% UA, 10 min | 70% EtOH with 1% UA, MW (250W) without vacuum, 40 s | 50% EtOH, 10min | ddH2O rinses × 2 | ddH2O rinses × 2 | 70% EtOH with 1% UA, MW (250W) without vacuum, 40 s | 70% EtOH with 1% UA, MW (250W) without vacuum, 40 s | 70% EtOH with 1% UA, MW (250W) without vacuum, 40 s | |||||
| 70% EtOH with 1% UA, 1 hr | 80% EtOH, 10 min | 50% acetone MW 40 s | 80% EtOH with 1% UA, 10 min | 90% EtOH with 1% UA, MW (250W) without vacuum, 40 s | 70% EtOH with 1% UA, 1 hr | 50% acetone MW 40 s | 50% acetone MW 40 s | 90% EtOH with 1% UA, MW (250W) without vacuum, 40 s | 90% EtOH with 1% UA, MW (250W) without vacuum, 40 s | 90% EtOH with 1% UA, MW (250W) without vacuum, 40 s s | |||||
| dehydrated further in EtOH and PO | 95% EtOH, 10 min | 70% acetone MW 40 s | 95% EtOH with 1% UA, 10 min | 100% EtOH with 1% UA, MW (250W) without vacuum, 40 s | dehydrated further in EtOH and PO | 70% acetone MW 40 s | 70% acetone MW 40 s | 100% EtOH with 1% UA, MW (250W) without vacuum, 40 s | 100% EtOH with 1% UA, MW (250W) without vacuum, 40 s | 100% EtOH with 1% UA, MW (250W) without vacuum, 40 s | |||||
| ↓ | 100% EtOH, 10 min × 4 | 90% acetone MW 40 s | 100% EtOH with 1% UA, 10 min | 100% EtOH, MW (250W) without vacuum, 40 s | ↓ | 90% acetone MW 40 s | 90% acetone MW 40 s | 100% EtOH, MW (250W) without vacuum, 40 s | 100% EtOH, MW (250W) without vacuum, 40 s | 100% EtOH, MW (250W) without vacuum, 40 s | |||||
| ↓ | PO, 15 min × 2 | 100% acetone MW 40 s | PO, 15 min | 1:1 EtOH to acetone, MW (250W) without vacuum, 40 s | ↓ | 100% acetone MW 40 s | 100% acetone MW 40 s | 1:1 EtOH to PO, 10 min | 1:1 EtOH to PO, 10 min | 1:1 EtOH to PO, 10 min | |||||
| ↓ | ↓ | ↓ | ↓ | 1:2 EtOH to acetone, MW (250W) without vacuum, 40 s | ↓ | ↓ | ↓ | 1:2 EtOH to PO, 10 min | 1:2 EtOH to PO, 10 min | 1:2 EtOH to PO, 10 min | |||||
| ↓ | ↓ | ↓ | ↓ | 100% acetone 3 times, MW (250W) without vacuum, 40 s | ↓ | ↓ | ↓ | 100% PO 3 times, 10 min ea | 100% PO 3 times, 10 min ea | 100% PO 3 times, 10 min ea | |||||
| infiltration | Epon (details unknown) | 1:1 PO to LX-112, 1 hr | 1:1 aceton to Epon/Spurr's, 1 hr, RT | 1:1 PO to LX-112, 1 hr | 1:1 acetone to LX-112, MW (250W) under vacuum, 3 min | Epon (details unknown) | 1:1 aceton to Epon/Spurr's, 1 hr, RT) under vacuum, 3 min | 1:1 aceton to Epon/Spurr's, 1 hr, RT | 1:1 PO to LX-112, 1 hr | 1:1 PO to LX-112, 1 hr | 1:1 PO to LX-112, 1 hr | ||||
| ↓ | 2:1 LX-112 with DMP-30 to PO, overnight | 1:2 acetone to Epon/Spurr's, overnight | 2:1 LX-112 with DMP-30 to PO, overnight | 1:4 acetone to LX-112, MW (250W) under vacuum, 3 min↓ | 1:2 acetone to Epon/Spurr's, overnight | 1:2 acetone to Epon/Spurr's, overnight1:2 PO to LX-112, overnight | 1:2 PO to LX-112, overnight | 1:2 PO to LX-112, overnight | |||||||
| ↓ | 100 % LX-112 with DMP-30, 2 hr | Epon/Spurr's several hr | 100 % LX-112 with DMP-30, 2 hr | LX-112, MW (250W) under vacuum, 3 min × 4 | ↓ | Epon/Spurr's several hr× 4 | Epon/Spurr's several hr | 100% LX-112, 3 times, 1 hr ea | 100% LX-112, 3 times, 1 hr ea | 100% LX-112, 3 times, 1 hr ea | |||||
| ↓ | Fresh LX-112 with DMP-30 under desicant for 1 hr at least.↓ | ↓ | ↓ | ↓ | ↓ | ↓ | ↓ | ↓ | |||||||
| resin polymerization | 60°C for 48 hr | 60°C for 48 hr | 60°C for 48 hr | 60°C for 48 hr | 60°C for 48 hr | 60°C for 48 hr | 60°C for 48 hr | 60°C for 48 hr | 60°C for 48 hr | ||||||
| post-section stain | ? | ethanolic UA, Lead citrate | ethanolic UA, Lead citrate | Lead citrate (series K24); ethanolic UA and lead citrate (series LRHXG and oblique) | ? | ethanolic UA, Lead citrate | ethanolic UA, Lead citrate | ethanolic UA, Lead citrate | ethanolic UA, Lead citrate | ethanolic UA, Lead citrate | aqueous UA, Lead citrate | aqueous UA, Lead citrate | EM | aqueous UA, Lead citrate | |
| EM | JEOL JEM-100B (seris K24); JEOL JEM-1230 (series LRHXG and oblique) | JEOL JEM-1200EX (Series UGXI); JEOL JEM-1230 (series NQDBP, ZSGCZ) | JEOL JEM -12301200EX | JEOL JEM-1230 | JEOL JEM-100B (seris K24); JEOL JEM-1230 (series LRHXG and oblique)JEOL JEM 1200EX | JEOL JEM-1230 | JEOL JEM-1230 | JEOL JEM-1230 (series); Zeiss Supra 40 (series) | Zeiss Supra 40 | ||||||
| imaging device/media | film (series UGXIK24); Gatan UltraScan4000 CCD camera (series NQDBP, ZSGCZ) | Gatan UltraScan4000 CCD camera | Gatan UltraScan4000 CCD camera | LRHXG and oblique) | film (series K24UGXI); Gatan UltraScan4000 CCD camera (series LRHXG and obliqueNQDBP, ZSGCZ) | film | Gatan UltraScan4000 CCD camera | Gatan UltraScan4000 CCD camera | Gatan UltraScan4000 CCD camera; transmitted electron detector | transmitted electron detector | Notes | The Sorra and Harris (1998) paper shows data from series UGXI. The series NQDBP and ZSGCZ were collected later. Data from series UGXI were also used in Kirov et al. (1999). | Gatan UltraScan4000 CCD camera | Gatan UltraScan4000 CCD camera; transmitted electron detector | transmitted electron detector |
| Notes | The Harris and Stevens (1989) paper shows data from series K24. The series "Oblique" (aka Rat34CA1BS12 1-91) and "Apical" (aka LRHXG, volumejosef, or Rat34CA1BS12 101-294) were collected later and used for the Harris et al. (2015) paper. All of these series are available at: https://neurodata.io/data/kharris15 Data from series LRHXG were also used in Bourne et al. (2007). | The Sorra and Harris (1998) paper shows data from series UGXI. The series NQDBP and ZSGCZ were collected later. Data from series UGXI were also used in Kirov et al. (1999). | Data from this series were also used in Bourne et al. (2007). | See Kuwajima et al (2013) for protcols for perfusion-fixation, tissue processing, and tSEM imaging. | See Kuwajima et al (2013) for protcols for perfusion-fixation, tissue processing, and tSEM imaging. |