Wholemount immunofluorescence staining, tissue clearing, and 3-D imaging (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Maho Shibata

Keywords

[‘3D imaging’, ‘Wholemount immunofluorescence’]

Subjects

[‘Imaging’]

Release Date

2024-10-05

Abstract

This protocol is used for wholemount staining of mouse anterior prostate. The tissue is dissected and fixed overnight, then stained wholemount for immunofluorescence. The tissue is cleared in Ce3D+ and imaged 3-D by confocal microscopy.

Procedure

Anterior prostates (AP) were dissected in 1X PBS. For prostates from mice 4 weeks and older, the lobes of the AP were split, and one half was used for whole-mount staining. The tissue was fixed overnight in 4% paraformaldehyde at 4C, then permeabilized with 2% Triton X-100 in 1x PBS for 3 hours on a rocker. All subsequent incubations were performed on a rocker at room temperature. The tissue was incubated with blocking buffer (10% FBS, 0.02% sodium azide, 1% BSA, 5% DMSO, and 0.2% Triton in 1X PBS) for 3 hours. Primary antibody was incubated overnight and washed in wash buffer (5% DMSO, 0.2% Triton in 1X PBS) before staining with secondary antibody overnight. Tissues were incubated with 5 μg/mL 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) at 1:1000 dilution in wash buffer for 3 hours, then tissue was cleared in Ce3D+ (Anderson et al., 2020; Li et al., 2019). Ce3D+ solution was prepared in advance in a 50 mL conical tube by adding 20 g Histodenz (final concentration 86% w/v; Sigma, D2158), 10.5 g of 40% N-Methylacetamide in 1X PBS (final concentration 22% w/v; Sigma, M26305), and 22.5 mg Triton-X-100 (final concentration 0.1% w/v) and mixing overnight on a rotator (Anderson, 2021). Stained tissue was incubated in Ce3D+ overnight, then cleared tissue was mounted onto a glass slide in the same Ce3D+ solution (Refractive index ND = 1.515) (Anderson et al., 2020). A silicon spacer was placed around the tissue in accordance with tissue thickness (200 μm spacer for 200 μm thick tissue), and the mounted tissue was sealed with a glass coverslip on top with nail polish along the edges. Images were acquired on Zeiss Cell Observer Spinning Disk and Zeiss 980 Confocal microscopes. Z stack images were acquired starting from the surface of the tissue. High resolution images of TUBB3-labeled nerve fibers and synaptophysin (SYP) puncta were acquired using Airyscan imaging on the Zeiss 980 Confocal microscope. Two-photon spectral imaging was performed on the Zeiss 980 Confocal microscope using 920nm laser excitation. Fluorophore emission curves were generated by probing a 32-channel lambda stack image set of the sample, and online linear spectral unmixing was conducted using Zen Software (Zeiss) to generate the spectral image.

References

1. Anderson, M. J., Magidson, V., Kageyama, R. and Lewandoski, M. (2020). Fgf4 maintains Hes7 levels critical for normal somite segmentation clock function. eLife 9.
2. Anderson, M. J., Magidson, V. and Lewandoski, M. (2021). Ce3D+ and Ce3D++ Tissue Clearing. Bio-protocol.
3. Li, W., Germain, R. N. and Gerner, M. Y. (2019). High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging. Nat Protoc 14, 1708-1733.

Consortium

GenitoUrinary Development Molecular Anatomy Project (GUDMAP) Consortium