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