Versions Compared

Old Version 23

changes.mady.by.user Masaaki Kuwajima

Saved on

compared with

New Version Current

changes.mady.by.user Masaaki Kuwajima

Saved on

Key

  • This line was added.
  • This line was removed.
  • Formatting was changed.

Table of Contents

This page can also be accessed with this URL: https://bit.ly/3yAutLp

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 [concentration (in mM): 2.0 CaCl2, 11.0 d-glucose
  • CA1 = hippocampal area CA1
    • SR = stratum radiatum
    • SLM = stratum lacunosum-moleculare
  • DG = hippocampal dentate gyrus
    • IML = inner molecular layer
    • MML = middle molecular layer
    • OML = outer molecular layer
  • EtOH = ethanol
  • KRC = Krebs–Ringer Carbicarb buffer
    • composition in mM: 118 NaCl, 4.7 KCl, 2.0 CaCl2, 4.0 MgSO4
    , 118 NaCl
    • , 12.5 Na2CO3, 12.5 NaHCO3, 11.0 d-glucose ; pH 7.4; osmolality, 300–330 mmol/kg
    ]
  • MW = microwave
  • PND = postnatal day
  • PO = propylene oxide
  • RT = room temperature

Protocols

Bourne 2007Harris and Stevens (1989);Kirov 199960 (4111:1 aceton to Epon/Spurr's, 1 hr, RTresin polymerizationethanolic UA, Lead citrate
Sorra and Harris, (1998Papers

Harris and Stevens (1989)

Bourne et al. (2007)

Mishchenko et al. (2010)

Kinney et al. (

2013)

Bartol et al. (2015)

Harris et al. (2015)

Harris et al. (

2022; CA1-SR)

Bourne

Sorra and Harris, (

2011

1998)

dentate gyrusSLM3DEM SeriesUGXI, NQDBP, ZSGCZHZKDGGWPMS (also HCSBR and HWVKW?)

"Spine" (aka K24), "Oblique" (aka Rat34CA1BS12 1-91), "Apical" (aka LRHXG, volumejosef, or Rat34CA1BS12 101-294)

WRPVRat85LMFHLTD, FWNGV, FPNCT, XRZCT
AnimalsKari68, Kari69JB072JB031 (also JB037 and JB047?)Rat34Rat84RRat85JB023, JB024,BLE5, LED50, LED56, LE102, LE108, LE113MK01, LE108
SpeciesRatRatRatRatRatRatRatRatRat
StrainLong EvansLong EvansLong EvansLong EvansLong EvansLong EvansLong EvansLong EvansLong Evans
Age (PND)50-606556-64776868

Kirov et al. (1999)

Harris et al. (2022; CA1-SR)

Kirov et al. (1999)

Bourne et al. (2007)

Harris et al. (2022; CA1-SR)

Ostroff et al. (2002)


Bourne et al. (2007)

Harris et al. (2022; CA1-SR)

Bourne et al. (2007)

Harris et al. (2022; CA1-SR)

PapersHarris et al. (2022; CA1-SR)

Bourne and Harris (2011)

Bourne et al. (2013)

Bell et al. (2014)

Smith et al. (2016)

Chirillo et al. (2019)


Watson et al. (2016)

Bromer et al. (2018)

Harris et al. (2022; DG)

Samavat et al. (2023)

Harris et al. (2022; CA1-SLM)
3DEM Series

"Spine" (aka K24), "Oblique" (aka Rat34CA1BS12 1-91), "Apical" (aka LRHXG, volumejosef, or Rat34CA1BS12 101-294)

UGXI, NQDBP, ZSGCZWRPV
HZKDGGWPMS (also HCSBR and HWVKW?)3DEM SeriesRat85LMFHLTD, FWNGV, FPNCT, XRZCT
SRQHN, CSKBZ, CLZBJ, TNKPS, WHKYG, JNBMM, MPLTJ, RLCVK, NDKZB, MFBCF, RJZQR, MCZJJ, RFHTC, DTZVX, KSGRS, BBCHZ, YPMYD, BRNLG, DRZNC, BLSNK, HLWLQ, JHHZS, LFLNP, TYLYL, HNVRR, YSJNL, ZGBJY, JJPSC, QRFNB, HVCBQ, GFBZX, XGDJF, ZBZFH, CRQCR, RSDCP, VGQNSVRJXH, KHZRL, KCPXD
AnimalsRat34Kari68, Kari69Rat84R
JB072JB031 (also JB037 and JB047?)AnimalsRat85JB023, JB024,Rat88, Rat89BLE5, LED50, LED56, LE102, LE108, LE113MK01, LE108
SpeciesRatRatRatRatRatRatSpeciesRatRatRatRatRat
StrainLong EvansLong EvansLong EvansLong EvansLong EvansLong EvansStrainLong EvansLong EvansLong EvansLong EvansLong Evans
Age (PND)7750-6068156556-64Age (PND)6860 (JB023); 61 (JB024)15185 (BLE5); 179 (LED50); 121 (LED56); 146 (LE102); 170 (LE108); 150 (LE113)162 (MK01); 170 (LE108);
SexmalemalemalemalemalemaleSexmalemalemalemalemale
body weight (g)310279 (Kari68); 236 (Kari69)411
333263 (JB031); 361 (JB037); 365 (JB047)310body weight (g)337323 (JB023); 319 (JB024);
490 (BLE5); 623 (LED50); 448 (LED56); 540 (LE102); 648 (LE108); 490 (LE113)648 (LE108);
anesthesiapentobarbital 80mg/kg (ip)rapid decapitationpentobarbital 80mg/kg (ip)
halothanehalothaneanesthesiapentobarbital 80mg/kg (ip)halothanepentobarbital 80mg/kgpentobarbital 80mg/kghalothanehalothanehalothaneslice preparation/kg (ip)halothanehalothane
slice preparationN/Adissection in ice-cold ACSF; recovered in ACSF at 30-31°C for at least 1 hr before recordingsN/Adissection in ice-cold ACSF; recovered in ACSF at 30-31°C for at least 1 hr before recordingsdissection at RT; recovered in ACSF at 31°C before electrophysiology recordingsdissection at RT; recovered in ACSF at 31°C before electrophysiology recordingsN/Aslice preparationN/AN/Adissection at RT; recovered in ACSF at 31°C before electrophysiology recordingsN/Ain vivo electrophysiologyN/A
total time (min) in recording chamberN/A340 (Kari68); 270  (Kari69)N/A
260285 (JB031); 235  (JB037); 285 (JB047)(JB037); 285 (JB047)total time (min) in recording chamberN/A315 (JB023); 360 (JB024);N/Ain vivo electrophysiologyN/AN/A315 (JB023); 360 (JB024);N/AN/Atissue fixation
tissue fixationPerfused w/ 2% paraformaldehyde, 2.5% glutaraldehyde, 2 mM CaCl2 in 0.1 M sodium cacodylate buffer, pH 7.35, 37°C, 4 psi pressure2% paraformaldehyde, 6% glutaraldehyde, and 2 mM CaCl2 in 0.1 M sodium cacodylate buffer during 8 s MWPerfused w/ 2% paraformaldehyde, 6% glutaraldehyde, 2 mM CaCl2, 4 mM MgSO4 in 0.1 M cacodylate buffer, pH 7.4, 37°C, 4 psi pressure2% paraformaldehyde, 6% glutaraldehyde, and 2 mM CaCl2 in 0.1 M sodium cacodylate buffer during 8 10 s MW2% paraformaldehyde, 6% glutaraldehyde, in 0.1 M sodium cacodylate buffer with 1 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 MWtissue fixationPerfused 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 pressure2% paraformaldehyde, 6% glutaraldehyde, in 0.1 M sodium cacodylate buffer with 2 mM CaCl2, 4 mM MgSO4 during 8-20 s MWPerfused w/ 2% paraformaldehyde, 6% , 2.5% glutaraldehyde, 2 mM CaCl2, 4 mM MgSO4 in 0.1 M cacodylate buffer , pH 7.4, 37°C, 4 psi pressure(postfixed overnight in the same fixative, but with 6% glutaraldehyde)Perfused w/ KRC, followed by 2% paraformaldehydeformaldehyde, 6% 2.5% glutaraldehyde, 2 mM CaCl2, 4 mM MgSO4 in 0.1 M cacodylate buffer, pH 7.4, 37°C, 4 psi pressure2% paraformaldehyde, 6% glutaraldehyde, in 0.1 M sodium cacodylate buffer with 2 mM CaCl2, 4 mM MgSO4 during 8-20 s MW35, 37°C, 80-180 mmHg pressurePerfused 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 pressurePerfused w/ KRC, followed by 2% formaldehyde, 2.5% glutaraldehyde, 2 mM CaCl2, 4 mM MgSO4 in 80-180 mmHg pressure

0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer , pH 7.35, 37°C, 120 mmHg pressurewashes0.1 M sodium cacodylate buffer washes
0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washes
0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washes
reduced osmium1.5% K4Fe(CN)6, 1 % OsO4 in 0.1 M sodium cacodylate buffer washes, 1 hr0.1 M sodium cacodylate buffer washes1.5% K4Fe(CN)6, 1 % OsO4 in 0.1 M sodium cacodylate buffer washes, 1 hr0.1 M sodium cacodylate buffer washes1.5% K4Fe(CN)6 and 1% OsO4 in 0.1 M sodium cacodylate buffer washesreduced osmium, cool to below 15°C and MW 2.5 min.1.5% K4Fe(CN)6, 1 % OsO4 in 0.1 M sodium cacodylate buffer, 1 hr1.5% K4Fe(CN)6, 1 % OsO4 in 0.1 M sodium cacodylate buffer, 1 hr1.5% K4Fe(CN)6, 1% OsO4 in 0.1 M sodium cacodylate buffer, MW (175 W) under vacuum, 1 min on - 1 min off - 1 min on. Cool to below 15°C and repeat.1.5% K4Fe(CN)6, 1 % OsO4 in 0.1 M sodium cacodylate buffer, 1 hr1.5% K4Fe(CN)6 and 1% OsO4 in 0.1 M sodium cacodylate buffer, cool . Cool to below 15°C and MW 2.5 min.repeat.reduced osmium1.5% 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 washes0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washes
0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washes
0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washesbuffer washes
osmium1% OsO4, 1 hr1% OsO4, 1 hr1% OsO4 in 0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washesosmium, cool to below 15°C and MW 2.5 min.1% OsO4, 1 hr1% OsO4, 1 hr1% OsO4 in 0.1M sodium cacodylate buffer, MW (175 W) under vacuum. 1 min on - 1 min off - 1 min on. Cool to below 15°C and repeat.1% OsO4, 1 hr1% OsO4 in 0.1 M sodium cacodylate buffer, cool to below 15°C and MW 2.5 min.repeat.osmium1% 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 washes0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washes
0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washes
0.1 M sodium cacodylate buffer washes0.1 M sodium cacodylate buffer washes


ddH2O rinsesddH2O rinsesddH2O rinsesddH2O rinsesddH2O rinses
ddH2O rinsesddH2O rinses
ddH2O rinsesddH2O rinses
dehydration & UA30% EtOH, 10 min50% EtOH, 10 min 1% UA (aq), cooled on ice, MW 2.5 min
50% EtOH with 1% UA, 10 min50% EtOH with 1% UA, MW (250W) without vacuum, 40 s 30% EtOH, 10 min1% UA (aq), cooled on ice, MW 2.5 min, 40 s dehydration & UA1% UA (aq), cooled on ice, MW 2.5 min50% 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, 10min70% EtOH with 1% UA, 1 hrddH2O rinses × 2
70% EtOH with 1% UA, 10 min70% EtOH with 1% UA, MW (250W) without vacuum, 40 s 50% EtOH, 10minddH2O rinses × 2
ddH2O rinses × 270% 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 hr80% EtOH, 10 min 50% acetone MW 40 s
80% EtOH with 1% UA, 10 min90% EtOH with 1% UA, MW (250W) without vacuum, 40 s 70% EtOH with 1% UA, 1 hr50% acetone MW 40 s40 s 
50% acetone MW 40 s90% 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 

dehydrated further in EtOH and PO95% EtOH, 10 min 70% acetone MW 40 s
95% EtOH with 1% UA, 10 min100% EtOH with 1% UA, MW (250W) without vacuum, 40 s dehydrated further in EtOH and PO
70% acetone MW 40 s70% acetone MW 40 s100% 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 × 490% acetone MW 40 s
100% EtOH with 1% UA, 10 min100% EtOH, MW (250W) without vacuum, 40 s ↓90% acetone MW 40 s
90% acetone MW 40 s100% 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 × 2100% acetone MW 40 s
PO, 15 min1:1 EtOH to acetone, MW (250W)  without vacuum, 40 s ↓100% acetone MW 40 s
100% acetone MW 40 s1:1 EtOH to PO, 10 min
1:1 EtOH to PO, 10 min1:1 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 min1:2 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 ea100% PO 3 times, 10 min ea
resin infiltrationEpon (details unknown)1:1 PO to LX-112, 1 hr1:1 aceton to Epon/Spurr's, 1 hr, RT
1:1 PO to LX-112, 1 hr1:1 acetone to LX-112, MW (250W) under vacuum, 3 minEpon (details unknown)

resin infiltration

1:1 aceton to Epon/Spurr's, 1 hr, RT1:1 PO to LX-112, 1 hr
1:1 PO to LX-112, 1 hr1:1 PO to LX-112, 1 hr

↓2:1 LX-112 with DMP-30 to PO, overnight1:2 acetone to Epon/Spurr's, overnight
2:1 LX-112 with DMP-30 to PO, overnight1:4 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, overnight1:2 PO to LX-112, overnight

↓100 % LX-112 with DMP-30, 2 hrEpon/Spurr's several hr
100 % LX-112 with DMP-30, 2 hrLX-112, MW (250W) under vacuum, 3 min × 4↓Epon/Spurr's several hr
Epon/Spurr's several hr100% LX-112, 3 times, 1 hr ea
100% LX-112, 3 times, 1 hr ea100% LX-112, 3 times, 1 hr ea

↓Fresh LX-112 with DMP-30 under desicant for 1 hr at least.↓
↓↓
↓↓
↓↓↓
resin polymerization60°C for 48 hr60°C for 48 hr60°C for 48 hr60°C for 48 hr60°C for 48 hr60°C for 48 hrresin polymerization60°C for 48 hr60°C for 48 hr
60°C for 48 hr60°C for 48 hr
post-section stain?ethanolic UA, Lead citrateLead citrate (series K24); ethanolic UA and lead citrate (series LRHXG and oblique)?ethanolic UA, Lead citrate
ethanolic UA, Lead citrateethanolic UA, Lead citratepost-section stainethanolic UA, Lead citrateethanolic UA, Lead citrate
aqueous UA, Lead citrateaqueous UA, Lead citrateaqueous UA, Lead citrateEM
EMJEOL JEM-100B (seris K24); JEOL JEM-1230 (series LRHXG and oblique)JEOL JEM-1200EX (Series UGXI); JEOL JEM-1230 (series NQDBP, ZSGCZ)JEOL JEM -12301200EXJEOL JEM -12301200EXJEOL JEM-100B (seris K24); 1230JEOL JEM-1230 (series LRHXG and oblique)JEOL JEM 1200EXEMJEOL JEM-1230JEOL JEM-1230
JEOL JEM-1230 (series); Zeiss Supra 40 (series)Zeiss Supra 40
imaging device/mediafilm (series K24); Gatan UltraScan4000 CCD camera (series LRHXG and oblique)film (series UGXI); Gatan UltraScan4000 CCD camera (series NQDBP, ZSGCZ)Gatan UltraScan4000 CCD camerafilmfilmGatan UltraScan4000 CCD camerafilm (series K24); Gatan UltraScan4000 CCD camera (series LRHXG and oblique)filmimaging device/mediaGatan UltraScan4000 CCD cameraGatan UltraScan4000 CCD camera
Gatan UltraScan4000 CCD camera; transmitted electron detectortransmitted electron detectorNotes

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).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 and  BossDB.

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).


Notes


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.