A compilation of previously used tissue processing protocols

Owned by Masaaki Kuwajima
Last updated: Jun 26, 2024
8 min read

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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
  • 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, 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

Papers

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)

Sorra and Harris, (1998)

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)body 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/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 recordingsslice preparationN/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)total time (min) in recording chamberN/A315 (JB023); 360 (JB024);N/Ain vivo electrophysiologyN/A
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 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, 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, in 0.1 M sodium cacodylate buffer with 2 mM CaCl2, 4 mM MgSO4 during 8-20 s MWPerfused w/ 2% paraformaldehyde, 2.5% glutaraldehyde, 2 mM CaCl2, 4 mM MgSO4 in 0.1 M cacodylate buffer (postfixed overnight in the same fixative, but with 6% glutaraldehyde)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, 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, 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 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
reduced osmium1.5% 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 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, 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.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 washes
osmium1% OsO4, 1 hr1% OsO4, 1 hr1% OsO4 in 0.1 M sodium cacodylate buffer, 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.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 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 
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 
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 
70% 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 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 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 min

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, 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 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 stainLead 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 citrate
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 1200EXJEOL JEM 1200EXJEOL JEM-1230JEOL JEM-1230EMJEOL 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)filmfilmGatan UltraScan4000 CCD cameraGatan UltraScan4000 CCD cameraimaging device/mediaGatan UltraScan4000 CCD cameraGatan UltraScan4000 CCD camera
Gatan UltraScan4000 CCD camera; transmitted electron detectortransmitted 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.



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