TABLE 1.

Studies that applied different 3D visualization and analysis approaches in mouse ovaries.

	Clearing reagent	Stage	Fixative	Immunolabeling applied (Y/N)	Microscope used	Structures analyzed	Quantitative analysis	References
Organic solvent based	BABB	Embryonic, postnatal, and adult	4% PFA	Yes	Confocal microscope and optical projection tomography	3D geography of lymphatic and blood vessels	N/A	Svingen et al., 2012
	BABB	Embryonic	4% PFA	Yes	Confocal microscope	Spatiotemporal characteristics of meiotic initiation in germ cells	Imaris and Matlab	Soygur et al., 2021
	BABB	Postnatal	4% PFA and 1% Glut	No (stained with a fluorescent dye, LysoTracker)	Confocal microscope	Apoptotic granulosa cells in follicles	N/A	Zucker and Jeffay, 2006
	BABB	Postnatal and adult	MeOH:DMSO	Yes	Confocal microscope	The numbers and spatial distribution of follicles	Volocity and Matlab	Faire et al., 2015
	iDISCO	Adult	% PFA	Yes	Spinning disk confocal microscope	Growing follicles and interstitial compartment	N/A	McKey et al., 2020
Aqueous based	ScaleA2	Embryonic	% PFA	Yes	Confocal microscope	Total germ cell numbers	Imaris	Malki et al., 2015
	CUBIC	Embryonic	% PFA	Yes (endogenous mCerulean, mCherry, and mOrange are also visualized)	Confocal microscope	Synchrony of meiotic onset and cytoplasmic sharing in germ cell clones	Imaris	Soygur et al., 2021
	CUBIC	Postnatal and adult	% PFA	Yes	Light-sheet microscope	Follicle numbers, 3D modeling of ovarian innervation and vasculature	Imaris	Tong et al., 2020
	CUBIC	Adult	% PFA	No (endogenous GFP signal is visualized)	Confocal and light-sheet microscopes	3D visualization of all cells in the ovary	N/A	Kagami et al., 2018
	CUBIC	Adult	% PFA	Yes (endogenous Tomato signal is also visualized)	Light-sheet microscope	Growing follicles, vasculature, and interstitial compartment	N/A	McKey et al., 2020
Hydrogel embedding	CLARITY	Postnatal, adult, and aged	% PFA	Yes	Confocal microscope	Dynamics of follicles, 3D structure and role of vasculature	Imaris and Matlab	Feng et al., 2017
	CLARITY	Adult	% PFA	Yes	Light-sheet microscope	Follicle numbers and vasculature	Imaris	Ma et al., 2018
Combined	iDISCO and CUBIC	Adult	% PFA	Yes	Light-sheet microscope and spinning disk confocal microscope	Growing follicles, oocytes, vasculature, and interstitial compartment	N/A	McKey et al., 2020
	CLARITY and ScaleA2	Adult	% PFA	Yes (endogenous td Tomato signal is also visualized)	Light-sheet microscope	Blood and lymphatic vessels	Ilastik and Imaris	Oren et al., 2018

PFA, paraformaldehyde; Glut, glutaraldehyde; MeOH, methanol; DMSO, dimethyl sulfoxide; N/A, not applied.