Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods

Breast Cancer Research

Table 1 A comparison of selected clearing methods in the mammary gland

Method	Method overview	RI	Clearing capability	Duration^a	Preservation		Whole mount IHC	Long-term storage	Rehydration and sectioning	Reference
Method	Method overview	RI	Clearing capability	Duration^a	structure	FP	Whole mount IHC	Long-term storage	Rehydration and sectioning	Reference
Uncleared	No clearing protocol applied	−	−	1 day	Preserved	Preserved	Compatible	No	Possible^f	−
3DISCO	Organic solvent-based	1.56	Strong	2 days	Compromised	Rapid loss	Difficult^c	No	Not possible	[8]
PACT-RC	Aqueous solution-based (hydrogel embedding)	1.45	Weak	10 days	Preserved, mild expansion	Preserved	Compatible	Yes	Not possible	[11]
PACT-sRIMS	Aqueous solution-based (hydrogel embedding)	1.46	Weak	13 days	Preserved	Preserved	Compatible	No	Possible^f	[11]
CUBIC	Aqueous solution-based (simple immersion)	1.48–9	Strong	5 days	Preserved, mild expansion	Some loss^b	Semi-compatible^d	No	Confirmed	[10]
SeeDB	Aqueous solution-based (simple immersion)	1.49	Moderate	5 days	Preserved, mild shrinkage	Preserved	Compatible	No^e	Confirmed	[9]

^aDuration from the time of tissue harvest (includes fixation time typically 6–16 h for mammary tissue). ^bAs previously reported [10, 39] and the observed requirement for slightly higher laser power for confocal imaging. This may be improved by using the second generation protocol (using Reagent 1A). ^cUsing the three-dimensional imaging of solvent-cleared organs combined with optimised whole-mount immunolabelling procedures iDISCO/three-dimensional imaging of solvent-cleared organs (3DISCO) method. The fluorescence signal is rapidly quenched using benzyl alcohol benzyl benzoate (BABB) and a specialised imaging chamber is required for dibenzyl ether (DBE). ^dSome antibodies (i.e. E-cadherin, K8 and human epidermal growth factor receptor 2) do not perform as well as in other clearing protocols. ^eOptimally imaged within 2 weeks but may be stored for several months. ^fLikely to be compatible, but not tested in this study. RI refractive index, FP fluorescent protein, IHC immunohistochemical analysis, PACT-RC passive clarity technique-Rapiclear, sRIMS sorbitol refractive index matching solution, CUBIC clear unobstructed brain imaging cocktails, SeeDB see deep brain

ISSN: 1465-542X