A compilation of previously used tissue processing protocols

1 Abbreviations
2 Protocols

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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)	Papers	Harris 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) Nam et al. (2025)	Harris et al. (2022; CA1-SLM)

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)	Papers	Harris 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) Nam et al. (2025)	Harris et al. (2022; CA1-SLM)
3DEM Series Codes	"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?)	3DEM Series	Rat85LM	FHLTD, 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, VGQNS	VRJXH, KHZRL, KCPXD
Animals	Rat34	Kari68, Kari69	Rat84R		JB072	JB031 (also JB037 and JB047?)	Animals	Rat85	JB023, JB024,	Rat88, Rat89	BLE5, LED50, LED56, LE102, LE108, LE113	MK01, LE108
Species	Rat	Rat	Rat	Rat	Rat	Rat	Species	Rat	Rat	Rat	Rat	Rat
Strain	Long Evans	Long Evans	Long Evans	Long Evans	Long Evans	Long Evans	Strain	Long Evans	Long Evans	Long Evans	Long Evans	Long Evans
Age (PND)	77	50-60	68	15	65	56-64	Age (PND)	68	60 (JB023); 61 (JB024)	15	185 (BLE5); 179 (LED50); 121 (LED56); 146 (LE102); 170 (LE108); 150 (LE113)	162 (MK01); 170 (LE108);
Sex	male	male	male	male	male	male	Sex	male	male	male	male	male
body weight (g)	310	279 (Kari68); 236 (Kari69)	411		333	263 (JB031); 361 (JB037); 365 (JB047)	body weight (g)	337	323 (JB023); 319 (JB024);		490 (BLE5); 623 (LED50); 448 (LED56); 540 (LE102); 648 (LE108); 490 (LE113)	648 (LE108);
anesthesia	pentobarbital 80mg/kg (ip)	rapid decapitation	pentobarbital 80mg/kg (ip)		halothane	halothane	anesthesia	pentobarbital 80mg/kg (ip)	halothane	pentobarbital 80mg/kg (ip)	halothane	halothane
slice preparation	N/A	dissection in ice-cold ACSF; recovered in ACSF at 30-31°C for at least 1 hr before recordings	N/A	dissection in ice-cold ACSF; recovered in ACSF at 30-31°C for at least 1 hr before recordings	dissection at RT; recovered in ACSF at 31°C before electrophysiology recordings	dissection at RT; recovered in ACSF at 31°C before electrophysiology recordings	slice preparation	N/A	dissection at RT; recovered in ACSF at 31°C before electrophysiology recordings	N/A	in vivo electrophysiology	N/A
total time (min) in recording chamber	N/A	340 (Kari68); 270 (Kari69)	N/A		260	285 (JB031); 235 (JB037); 285 (JB047)	total time (min) in recording chamber	N/A	315 (JB023); 360 (JB024);	N/A	in vivo electrophysiology	N/A
tissue fixation	Perfused w/ 2% paraformaldehyde, 2.5% glutaraldehyde, 2 mM CaCl₂ in 0.1 M sodium cacodylate buffer, pH 7.35, 37°C, 4 psi pressure	2% paraformaldehyde, 6% glutaraldehyde, and 2 mM CaCl₂ in 0.1 M sodium cacodylate buffer during 8 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, and 2 mM CaCl₂ in 0.1 M sodium cacodylate buffer during 10 s MW	2% paraformaldehyde, 6% glutaraldehyde, in 0.1 M sodium cacodylate buffer with 1 mM CaCl₂ and 2 mM MgCl₂ during 8-20 s MW	2% paraformaldehyde, 6% glutaraldehyde, in 0.1 M sodium cacodylate buffer with 1 mM CaCl₂ and 2 mM MgCl₂ during 8-20 s MW	tissue fixation	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/ 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 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, 80-180 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	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, 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 hr	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, MW (175 W) under vacuum, 1 min on - 1 min off - 1 min on. Cool to below 15°C and repeat.	reduced osmium	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	0.1 M sodium cacodylate buffer washes
osmium	1% OsO₄, 1 hr	1% OsO₄, 1 hr	1% OsO₄ in 0.1 M sodium cacodylate buffer, cool to below 15°C and MW 2.5 min.	1% OsO₄, 1 hr	1% OsO₄, 1 hr	1% 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.	osmium	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	0.1 M sodium cacodylate buffer washes
		ddH₂O rinses	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	dehydration & UA	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		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		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
	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		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	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	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
resin 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	resin infiltration	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 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	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	resin polymerization	60°C for 48 hr	60°C for 48 hr		60°C for 48 hr	60°C for 48 hr
post-section stain	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	post-section stain	ethanolic UA, Lead citrate	ethanolic UA, Lead citrate		aqueous UA, Lead citrate	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 1200EX	JEOL JEM 1200EX	JEOL JEM-1230	JEOL JEM-1230	EM	JEOL JEM-1230	JEOL JEM-1230		JEOL JEM-1230 (series); Zeiss Supra 40 (series)	Zeiss Supra 40
imaging device/media	film (series K24); Gatan UltraScan4000 CCD camera (series LRHXG and oblique)	film (series UGXI); Gatan UltraScan4000 CCD camera (series NQDBP, ZSGCZ)	film	film	Gatan UltraScan4000 CCD camera	Gatan UltraScan4000 CCD camera	imaging device/media	Gatan UltraScan4000 CCD camera	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 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.